Quaternary nitrogen heterocyclic compounds for detecting aqueous monosaccharides in physiological fluids

ABSTRACT

Quaternary nitrogen heterocyclic boronic acid-containing compounds are described, which are sensitive to glucose and fructose, as well as a variety of other physiologically important analytes, such as aqueous chloride and iodide, and a method of using the compounds. Also disclosed is a contact lens doped with the quaternary nitrogen heterocyclic boronic acid-containing compound, and a method of using the doped contact lens to measure the concentration of analyte in tears under physiological conditions.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to fluorophores and more particularly to highlyfluorescent and analyte sensitive boronic acid containing fluorophoresand for methods of using same for measuring analyte concentrations, suchas glucose in physiological fluids, such as the blood and tears, in acontinuous and non-invasive manner. The invention further relates toophthalmic devices comprising the fluorophores, which interact with theanalyte to be measured providing an optical signal being indicative ofthe analyte level in an ocular fluid.

2. Background of the Related Art

Individuals suffering from diabetes mellitus have an abnormally highblood sugar level, generally because the pancreas does not secretesufficient amounts of the active hormone insulin into the bloodstream toregulate carbohydrate metabolism. If an abnormally high blood sugarlevel, known as a hyperglycemic condition, is allowed to continue forprolonged periods, the individual will suffer from the chroniccomplications of diabetes, including retinopathy, nephropathy,neuropathy and cardiovascular disease. Presently, approximately 150million people worldwide are affected by diabetes. Studies indicate thatdiabetic patients who are able to maintain near normal glycemic controlgreatly reduce the likelihood of these direct complications. Therefore,several tests have been developed to measure and control the glycemiccondition.

One common medical test to control glycemic condition is the directmeasurement of blood glucose levels. Blood glucose levels fluctuatesignificantly throughout a given day, being influenced by diet,activity, and treatment. Depending on the nature and severity of theindividual case, some patients must measure their blood glucose levelsup to seven times a day. Methods of glucose analysis includeelectrochemistry, near infrared spectroscopy, optical rotation,colorimetry, fluorimetry, and the enzyme-based method, the latter beingthe most commonly used. Unfortunately, the enzyme-based method hasseveral disadvantages, including the requirement of “finger pricking,”which is highly invasive and often inconvenient. It is known that manydiabetic patients often skip the analysis step, i.e., drawing blood, andadminister an estimated dose of insulin, which can lead to substantialfluctuations in insulin levels over time. Further, the enzyme-basedmethod is not continuous, thus putting the patient at risk ofunacceptably high or low glucose levels.

In recent years, various non-invasive and minimally-invasivetechnologies have been proposed in the academic and patent literature tomonitor glucose levels in the blood, ocular fluid, e.g., tears, aqueoushumor or interstitial fluid. For example, the GlucoWatch® non-invasivelymonitors glucose levels in the interstitial fluid every ten minutes forup to thirteen hours. However, the GlucoWatch® manufacturers expresslystate that the GlucoWatch® is designed to merely supplement conventionalblood glucose monitoring.

U.S. Pat. No. 6,681,127 discloses an ophthalmic lens, including achemical sensor, to determine the amount of an analyte, e.g., glucose,in an ocular fluid. Such ophthalmic lens includes a receptor moiety,which can bind either a specific analyte, e.g., glucose, or a detectablylabeled competitor moiety. The amount of detectably labeled competitormoiety which is displaced from the receptor moiety by the analyte ismeasured and provides a means of determining analyte concentration inthe ocular fluid. A disadvantage of this method includes the potentialthat other compounds are present in the fluid that are capable ofdisplacing the competitor moiety, thereby giving a false analyteconcentration.

It is well known in the glucose monitoring arts that tear glucose levelsdirectly track blood glucose levels, however, the concentration ofglucose in tears, e.g., 50-500 μM, is about ten times lower than thecorresponding blood glucose level (Van Haeringen, N. J., Surv.Ophthalmol., 29(2), 84-96 (1981); Gasser, A. R., et al., Am. J.Ophthalmol., 65(3), 414-420 (1968); Das, B. N., et al., J. Indian Med.Assoc., 93(4), 127-128 (1995); Chen, R., et al., J. CapillaryElectrophor., 3(5), 243-248 (1996); Perez, S. A., Electrophoresis,17(2), 352-358 (1996); Jin, Z., Anal. Chem., 69(7), 1326-1331 (1997)).Accordingly, to determine the concentration of glucose in tears requiresa methodology that is highly sensitive relative to standard bloodglucose methods. To date, attempts to monitor tear glucoseconcentrations have been invasive and applied non-continuousmethodologies.

Therefore, there is a continuing need for new methods of determinationof monosaccharide, e.g., glucose and fructose that are sensitive enoughto quantitatively determine monosaccharide levels in tears and otherbodily fluids under physiological conditions. These methods should becontinuous, non-invasive and uncomplicated, thereby ensuring thediabetic actively monitors their blood glucose levels.

Correspondingly, there is a need for methods of determination of levelsof a variety of other analytes in tears and other bodily fluids underphysiological conditions, for applications including monitoring ofpatient stability, medication compliance, exposure of individuals toenvironmental contaminants and toxins, etc.

SUMMARY OF THE INVENTION

The present invention generally relates to highly fluorescent andglucose sensitive boronic acid containing fluorophores which aresensitive to glucose and fructose, as well as a variety of otherphysiologically important analytes, such as aqueous chloride, iodide,fluoride and cynanide; and methods of using such fluorophores compounds.Preferably, the highly fluorescent and glucose sensitive boronic acidcontaining fluorophores comprise quaternary nitrogen heterocyclicboronic acid-containing compounds. Further, the present inventionrelates to using these sensitive fluorophores in glucose sensingophthalmic devices, e.g., off-the-shelf disposable plastic contactlenses that are coated or impregnated with novel glucose sensitivefluorophores.

In one aspect the present invention relates to novel quaternary nitrogenheterocyclic boronic acid-containing compounds including:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups.

In yet another aspect, the present invention relates to an opticaldevice, wherein the optical device comprises at least one fluorophore,wherein the fluorophore comprises a boronic acid group and anelectron-donor group and wherein the boronic acid group acts as anelectron-withdrawing group until interaction with a sugar therebycausing a decrease in the pK_(a) of the boronic acid and spectralchanges due to reduced charge-transfer. Preferably, the fluorophore is aquaternary nitrogen heterocyclic boronic acid-containing compoundincluding:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups.

Additional fluorophores that exhibit the necessary reduced chargetransfer spectral change include:

Preferably, the optical device is a contact lens that is used to measurethe concentration of glucose in tears under physiological conditions,wherein the contact lens includes at least one of the followingcompounds:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups;

Preferably, the analyte is glucose, wherein the concentration of glucosein the tear fluid is in the range from about 50 um to about 500 um.

In still another aspect, the present invention relates to a method ofmeasuring the concentration of an analyte in a physiological fluid, saidmethod comprising:

(a) contacting a fluorescence compound selected from the groupconsisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups,with the physiological fluid for sufficient time to at least partiallyinteract or react with the analyte; and

(b) measuring continuously the optical signal of the fluorescencecompound in the presence of the analyte for a sufficient time todetermine the concentration of analyte in the physiological fluid.

In the alternative, the quaternary nitrogen heterocyclic boronicacid-containing fluorophores may be used for analysis of other analytes,including but not limited to fluoride, chloride, iodide and cynanide.

In yet another aspect, the present invention relates to an ophthalmicsensor comprising:

a polymer matrix that accepts a sufficient amount of a fluorescencecompound within at least the outer surfaces of the polymer matrix,wherein the fluorescence compound interacts or reacts with an analyte toprovide an optical signal which is indicative of the analyteconcentration in an ocular fluid and wherein the fluorescence compoundis at least one member selected from the group consisting of:

-   -   wherein X is chloride, bromide or iodide and R is selected from        the group consisting of H, straight chain or branched C₁-C₄        alkyl group, C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano,        sulfonyl, and NR¹R², wherein R¹ and R² may be the same as or        different from one another and is independently selected from        the group consisting of H and C₁-C₄ alkyl groups;

In a further aspect, the present invention relates to a method ofmeasuring the concentration of glucose in ocular fluid, said methodcomprising:

(a) contacting a fluorescence compound selected from the groupconsisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups,

with the ocular fluid for sufficient time to at least partially interactor react with the glucose to provide an optical signal which isindicative of the glucose concentration in the ocular fluid.

The optical signal may include any change in fluorescence, such aschanges in fluorescence lifetime, intensity, emission maxima, absorptionmaxima, anisotropy and any measure of a parameter associated withfluorescence spectroscopy.

In another aspect, the present invention relates to including a secondsensing analyte compound to measure another analyte such as includingsensing compounds that measure the concentration of chlorides.

A further aspect of the invention relates to a compound selected fromthe group consisting of:

In another aspect, the invention relates to an analyte sensor comprisinga heterocyclic quaternary nitrogen compound containing at least oneheterocyclic quaternary ring nitrogen that is linked through a phenylring with a boronic acid group —B(OH)₂.

A further aspect of the invention relates to a method of determininglevel of an analyte at a locus containing or susceptible to presence ofsaid analyte, such method comprising exposing to said locus an analytesensor including a heterocyclic quaternary nitrogen compound containingat least one heterocyclic quaternary ring nitrogen that is linkedthrough a phenyl ring with a boronic acid group —B(OH)₂., anddetermining from an optical fluorescence signal of said heterocyclicquaternary nitrogen compound the level of the analyte at such locus.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWNGS

FIG. 1 shows the equilibrium reactions for the boronic acid/sugarinteraction.

FIG. 2 shows several novel quaternary nitrogen heterocyclic boronicacid-containing fluorophores.

FIG. 3A shows the absorption and emission spectra of o-BMOQBA(N-(2-boronobenzyl)-6-methoxyquinolinium bromide) in H₂O. The spectraare also representative of the respective m- and p-isomers and thecontrol compound BMOQ.

FIG. 3B shows the absorption and emission spectra of o-BMQBA(N-(2-boronobenzyl)-6-methylquinolinium bromide) in H₂O. The spectra arealso representative of the respective m- and p-isomers and the controlcompound BMQ.

FIG. 4 shows the emission spectra of o-BMQBA in pH 7.5 phosphate bufferwith (A) fructose, (B) glucose and (C) the intensity ratio at λ=427 nmin the absence, I′, and presence, I, of the sugar, respectively.

FIG. 5 shows the emission spectra of o-BMQBA in pH media with (A) 100 mMfructose, (B) 100 mM glucose and (C) buffer.

FIG. 6 shows the intensity ratio at λ=427 nm for o-BMQBA at specified pHvalues, I, relative to the intensity at pH 3.0, I_(o), (in the absenceof a sugar and in the presence of 100 mM of glucose or fructose).

FIG. 7 shows the emission spectra of o-BMQBA in (A) pH 5.0 buffer, (B)pH 6.0 buffer, (C) pH 7.0 buffer, and (D) pH 8.0 buffer at varyingglucose concentrations.

FIG. 8 shows the intensity ratio at λ=427 nm for o-BMQBA at varyingbuffered pH values and glucose concentrations at (A) high glucoseconcentrations and (B) low glucose concentrations (typical of thosefound in tears).

FIG. 9 shows the intensity ratio at λ=427 nm for o-BMQBA at varyingbuffered pH values and fructose concentrations at (A) high fructoseconcentrations and (B) low fructose concentrations.

FIG. 10A shows the emission spectra of o-BMQBA in pH 7.5 phosphatebuffer having 100 mM NaCl at varying fructose concentrations.

FIG. 10B shows the intensity ratio at λ=427 nm for o-BMQBA at specifiedfructose concentrations in the absence and presence of NaCl, where I′ isthe fluorescence intensity at 0 mM fructose and I is the correspondingintensity at the specified fructose concentration.

FIG. 10C shows the intensity ratio at λ=427 nm for o-BMQBA at lowfructose concentrations in the absence and presence of NaCl, where I′ isthe fluorescence intensity at 0 mM fructose and I is the correspondingintensity at the specified fructose concentration.

FIG. 11A shows the emission spectra of o-BMQBA in pH 7.5 phosphatebuffer having 100 mM NaCl at varying glucose concentrations.

FIG. 11B shows the intensity ratio at λ=427 nm for o-BMQBA at specifiedglucose concentrations in the absence and presence of NaCl, where I′ isthe fluorescence intensity at 0 mM glucose and I is the correspondingintensity at the specified glucose concentration.

FIG. 11C shows the intensity ratio at λ=427 nm for o-BMQBA at lowglucose concentrations in the absence and presence of NaCl, where I′ isthe fluorescence intensity at 0 mM glucose and I is the correspondingintensity at the specified glucose concentration.

FIG. 12 shows the emission spectra (λ_(ex)=345 nm) of o-BMOQBA in pH 7.5phosphate buffer with (A) glucose, (B) fructose and (C) the intensityratio at λ=450 nm in the absence, I′, and presence, I, of the sugar,respectively.

FIG. 13 shows the emission spectra (λ_(ex)=345 nm) of o-BMOQBA in pHmedia with (A) buffer, (B) 100 mM glucose, and (C) 100 mM fructose.

FIG. 14 shows the normalized intensity ratio at λ=450 nm (λ_(ex)=345 nm)for o-BMOQBA at specified pH values, wherein the normalized intensity isthe fluorescence intensity at the specified pH, I, relative to theintensity at pH 3.0, I_(o), (in the absence of a sugar and in thepresence of 100 mM of glucose or fructose).

FIG. 15 shows the emission spectra (λ_(ex)=345 nm) of o-BMOQBA in (A) pH5.0 buffer, (B) pH 6.0 buffer, (C) pH 7.0 buffer, and (D) pH 8.0 bufferat varying glucose concentrations.

FIG. 16 shows the intensity ratio at λ=450 nm (λ_(ex)=345 nm) foro-BMOQBA at varying buffered pH values and glucose concentrations at (A)high glucose concentrations and (B) low glucose concentrations (typicalof those found in tears).

FIG. 17 shows the intensity ratio at λ=450 nm (λ_(ex)=345 nm) foro-BMOQBA at varying buffered pH values and fructose concentrations at(A) high fructose concentrations and (B) low fructose concentrations.

FIG. 18A shows the emission spectra (λ_(ex)=345 nm) of o-BMOQBA in pH7.5 phosphate buffer having 100 mM NaCl at various fructoseconcentrations.

FIG. 18B shows the intensity ratio at λ=450 nm (λ_(ex)=345 nm) foro-BMOQBA at specified fructose concentrations in the absence andpresence of NaCl, where I′ is the fluorescence intensity at 0 mMfructose and I is the corresponding intensity at the specified fructoseconcentration.

FIG. 18C shows the intensity ratio at λ=450 nm (λ_(ex)=345 nm) foro-BMOQBA at low fructose concentrations in the absence and presence ofNaCl, where I′ is the fluorescence intensity at 0 mM fructose and I isthe corresponding intensity at the specified fructose concentration.

FIG. 19A shows the emission spectra (λ_(ex)=345 nm) of o-BMOQBA in pH7.5 phosphate buffer having 100 mM NaCl at various glucoseconcentrations.

FIG. 19B shows the intensity ratio at λ=450 nm (λ_(ex)=345 nm) foro-BMOQBA at specified glucose concentrations in the absence and presenceof NaCl, where I′ is the fluorescence intensity at 0 mM glucose and I isthe corresponding intensity at the specified glucose concentration.

FIG. 19C shows the intensity ratio at λ=450 nm (λ_(ex)=345 nm) foro-BMOQBA at low glucose concentrations in the absence and presence ofNaCl, where I′ is the fluorescence intensity at 0 mM glucose and I isthe corresponding intensity at the specified glucose concentration.

FIG. 20 shows the mean lifetime of o-BMOQBA at different buffered pHvalues.

FIG. 21 shows (A) the emission spectra of o-BMQBA leaching from aBMQBA-doped contact lens into a pH 7.5 buffer with time, (B) percent ofo-BMQBA remaining in the BMQBA-doped contact lens over time.

FIG. 22 shows the absorption and emission spectra of a o-BMQBA-dopedcontact lens in a pH 7.5 buffer.

FIG. 23A shows the emission spectra of a o-BMQBA-doped contact lens inpH 7.5 phosphate buffer with increasing concentrations of fructose.

FIG. 23B shows the intensity ratio of a o-BMQBA-doped contact lens in pH7.5 buffer at λ=420 nm in the absence, I′, and presence, I, of fructose.

FIG. 23C shows the intensity ratio of a o-BMQBA-doped contact lens in pH7.5 buffer at λ=420 nm at low fructose concentrations in the absence,I′, and presence, I, of the fructose, respectively.

FIG. 24A shows the emission spectra of a o-BMQBA-doped contact lens inpH 7.5 phosphate buffer with increasing concentrations of glucose.

FIG. 24B shows the intensity ratio of a o-BMQBA-doped contact lens in pH7.5 buffer at λ=420 nm in the absence, I′, and presence, I, of theglucose, respectively.

FIG. 24C shows the intensity ratio of a o-BMQBA-doped contact lens in pH7.5 buffer at λ=420 nm at low glucose concentrations in the absence, I′,and presence, I, of the glucose, respectively.

FIG. 25 shows the comparison of the intensity ratios of a o-BMQBA-dopedcontact lens in pH 7.5 buffer at λ=420 nm to the solution-basedmeasurements in pH 7.5 buffer at λ=427 nm for (A) high concentrations offructose, (B) low concentrations of fructose, (C) high concentrations ofglucose, and (D) low concentrations of glucose.

FIG. 26 shows (A) the emission spectra of o-BMOQBA leaching from aBMOQBA-doped contact lens into a pH 7.5 buffer with time, (B) percent ofo-BMOQBA remaining in the BMOQBA-doped contact lens over time.

FIG. 27A shows the emission spectra of a o-BMOQBA-doped contact lens inpH 7.5 phosphate buffer with increasing concentrations of fructose.

FIG. 27B shows the emission spectra of a o-BMOQBA-doped contact lens inpH 7.5 phosphate buffer with increasing concentrations of glucose.

FIG. 28 shows the intensity ratio of a o-BMOQBA-doped contact lens in pH7.5 buffer at λ=420 nm in the absence, I′, and presence, I, of fructose(●) or glucose (▪).

FIG. 29 shows the comparison of the intensity ratios of a o-BMOQBA-dopedcontact lens in pH 7.5 buffer at λ=420 nm to the solution-basedmeasurements in pH 7.5 buffer at λ=427 nm for (A) high concentrations ofglucose, and (B) low concentrations of glucose.

FIG. 30A shows the mean lifetime of the o-BMOQBA-doped contact lens withvarying chloride concentrations.

FIG. 30B shows the lifetime ratio of the o-BMOQBA-doped contact lenswith varying chloride concentrations, where τ′ represents the lifetimeat 0 mM chloride and π represents the lifetime at the specified chlorideconcentration.

FIG. 31 shows several boronic acid-containing fluorophores including:the stilbenes 4′-dimethylaminostilbene-4-boronic acid (DSTBA) and4′-cyanostilbene-4-boronic acid (CSTBA); the polyene1-(p-boronophenyl)-4-(p-dimethylaminophenyl)buta-1,2-diene (DDPBBA); andthe chalcones3-[4′-(dimethylamino)phenyl]-1-(4′-boronophenyl)-prop-2-en-1-one(Chalc 1) and5-[4′-(dimethylamino)phenyl]-1-(4′-boronophenyl)-pent-2,4-dien-1-one(Chalc 2).

FIG. 32A shows the emission spectra (λ_(ex)=340 nm) of DSTBA in pH 8.0buffer/methanol (2:1) with increasing concentrations of fructose.

FIG. 32B shows the emission spectra (λ_(ex)=320 nm) of CSTBA in pH 8.0buffer/methanol (2:1) with increasing concentrations of fructose.

FIG. 32C shows the ratiometric response of DSTBA to both fructose andglucose.

FIG. 32D shows the ratiometric response of CSTBA to both fructose andglucose.

FIG. 33A shows the emission spectra (λ_(ex)=340 nm) of DDPBBA in pH 8.0buffer/methanol (2:1) with increasing concentrations of fructose.

FIG. 33B shows the emission spectra (λ_(ex)=430 nm) of Chalc 2 in pH 8.0buffer/methanol (2:1) with increasing concentrations of fructose.

FIG. 34A shows the emission spectra (λ_(ex)=340 nm) of a DSTBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of glucose.

FIG. 34B shows the emission spectra (λ_(ex)=340 nm) of a DSTBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of fructose.

FIG. 34C shows the emission spectra (λ_(ex)=340 nm) of a CSTBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of glucose.

FIG. 34D shows the emission spectra (λ_(ex)=340 nm) of a CSTBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of fructose.

FIG. 35 shows the intensity ratio of a (A) DSTBA-doped contact lens and(B) CSTBA-doped contact lens in pH 8.0 buffer/methanol (2:1) in theabsence, I′, and presence, I, of sugar.

FIG. 36A shows the emission spectra (λ_(ex)=340 nm) of a DDPBBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of glucose.

FIG. 36B shows the emission spectra (λ_(ex)=340 nm) of a DDPBBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of fructose.

FIG. 36C shows the intensity ratio of a DDPBBA-doped contact lens in pH8.0 buffer/methanol (2:1) in the absence, I′, and presence, I, of sugar.

FIG. 37 shows the emission spectra of DDPBBA-doped contact lenses in pHmedia (buffer/methanol (2:1)) at (A) pH 9.0, (B) pH 8.0, (C) pH 7.0, and(D) pH 6.0.

FIG. 38A shows the emission spectra (λ_(ex)=460 nm) of a Chalc 2-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of glucose.

FIG. 38B shows the emission spectra (λ_(ex)=460 nm) of a Chalc 2-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of fructose.

FIG. 38C shows the intensity ratio of a Chalc 2-doped contact lens in pH8.0 buffer/methanol (2:1) in the absence, I′, and presence, I, of sugar.

FIG. 39 shows the emission spectra (λ_(ex)=360 nm) of compound (L) in pH7.0 buffer with glucose.

FIG. 40 shows the intensity ratio for response of compound (L) towardssugars in pH 7.0 phosphate buffer.

FIG. 41 shows the emission spectra (λ_(ex)=360 nm) of compound (M) in pH7.0 buffer with glucose.

FIG. 42 shows the intensity ratio for response of compound (M) towardssugars in pH 7.0 buffer.

FIG. 43 shows the emission spectra (λ_(ex)=345 nm) of compound (N) in pH7.0 buffer with glucose.

FIG. 44 shows the intensity ratio for response of compound (L) towardssugars in pH 7.0 buffer.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures are well known and commonlyemployed in the art. Conventional methods are used for these procedures,such as those provided in the art and various general references. Wherea term is provided in the singular, the inventors also contemplate theplural of that term. As employed throughout the disclosure, thefollowing terms shall be understood to have the following meanings.

“Biocompatible,” as used herein, refers to any material or a surface ofa material or an article which does not deteriorate appreciably and doesnot induce a significant immune response or deleterious tissue reaction,e.g., toxic reaction or significant irritation, over time when implantedinto or placed adjacent to the biological tissue of a subject.

An “ophthalmic device,” as used herein, refers to a contact lens (hardor soft), a corneal inlay, or implantable ophthalmic devices used in, onor about the eye or ocular vicinity.

An “ophthalmic sensor,” as used herein, comprises the molecular sensingmoiety and the ophthalmic device.

An “implantable ophthalmic device,” as used herein, refers to anophthalmic device, which is used in, on or about the eye or ocularvicinity. Exemplary implantable ophthalmic devices include, withoutlimitation, an intraocular lens, a subconjunctival lens, an intracorneallens, and a shunt or implant, e.g., a stent or a glaucoma shunt, thatcan rest on the cul de sac of an eye.

The term “contact lens,” as used herein, is intended to encompass anyhard or soft lens used on the eye or ocular vicinity for visioncorrection, diagnosis, sample collection, drug delivery, wound healing,cosmetic appearance, e.g., eye color modification, or other ophthalmicapplications. It can be a daily-disposable contact lens, a daily-wearcontact lens, or an extended-wear contact lens.

“Ophthalmically compatible,” as used herein, refers to a material orsurface of a material which may be in intimate contact with the ocularenvironment for an extended period of time without significantlymodifying the ocular environment.

“Ocular environment,” as used herein, refers to ocular fluids, e.g.,tear fluid, and ocular tissue, e.g., the cornea, and/or conjunctiva thatmay come into intimate contact with a contact lens.

“Fluorophore,” as used herein, is intended to encompass a chemical orbiochemical molecule or fragments thereof that is capable of interactingor reacting specifically with an analyte of interest in a sample toprovide one or more optical signals. Exemplary fluorophores includewithout limitation derivatives of phenyl boronic acid.

As used herein, “aryl” is intended to be broadly construed as referringto carbocyclic (e.g., phenyl, naphthyl), as well as heterocyclicaromatic groups (e.g., pyridyl, thienyl, furanyl, etc.), andencompassing unsubstituted as well as substituted aryl groups, whereinthe substituents of substituted aryl groups may include any stericallyacceptable substituents which are compatible with such aryl groups andwhich do not preclude the efficacy of the co-solvent compound for itsintended utility. Examples of substituents for substituted aryl groupsinclude one or more of halogen (e.g., fluoro, chloro, bromo, and iodo),amino, amido, C₁-C₄ alkyl, C₁-C₄ alkoxy, nitro, trifluoromethyl,hydroxy, hydroxyalkyl containing a C₁-C₄ alkyl moiety, etc.

“Changes in fluorescence,” as used herein, encompasses changes influorescence lifetime, intensity, emission maxima, absorption maxima,anisotropy, and any measurable parameter associated with fluorescencespectroscopy.

“Ratiometric sensing,” as used herein, encompasses comparativefluorescence intensities in the form of a ratio, whereby the numeratorand denominator were measured at the same emissive wavelength (if singleemission band) or different emissive wavelengths (if dual emission bandsor observed red or blue shifts).

Boronic acid molecular sensing moieties for sensing monosaccharides havebeen described in the literature (James, T. D., et al., Agnew. Chem.Int. Ed. Engl., 33, 2207 (1994); James, T. D., et al., J. Am. Chem.Soc., 117, 8982 (1995); Bielecki, M., et al., J. Chem. Soc. PerkinTrans., 2, 449 (1999); Dicesare, N., et al., Anal. Biochem., 294,154-160 (2001); Dicesare, N., et al., J. Photochem. Photobiol. A, 143,39-47 (2001); Dicesare, N., et al., Org. Lett., 3(24), 3891-3893 (2001);Dicesare, N., et al., Tetrahedron Lett., 43, 2615-2618 (2002)), thecontents of which are incorporated herein by reference for all purposes.

Boronic acid, —B(OH)₂ (represented by A in FIG. 1), is a weak Lewis acidwhich reversibly interacts with strong bases, e.g., hydroxyl groups,according to the reaction scheme shown in FIG. 1, to form anionicborates, —B(OH₃)⁻ (represented by B in FIG. 1). The pK_(a) of theboronic acid/anionic borate equilibrium is typically about 9.

Boronic acids also have a strong affinity for, and covalently bond with,diols, e.g., glucose, to form boronic acid diester groups (representedby C in FIG. 1), which reversibly interact with hydroxyl groups to formanionic boronate diester groups (represented by D in FIG. 1). The pK_(a)of the boronic acid diester/boronate diester equilibrium isapproximately 6, which is attributed to the increased Lewis acidity ofthe boronic acid diester complex. This large decrease in the pK_(a) ofthe boronic acid diester complex relative to the uncomplexed boronicacid permits the detection of sugars at neutral pH because substantialoptical changes are observed, e.g, the fluorescence intensity changeswith pH. However, if the pH of the bodily fluid environment is less thanneutral, the boronic acid molecule is preferably modified with a groupsuch as an electron withdrawing or donation group to increasesensitivity to sugars in a lower pH environment.

The invention described herein generally relates to novel fluorophoresensing moieties for detecting/measuring analytes, in particularglucose, in a body fluid and a method for using said novel molecularsensing moieties. The fluorophore sensing moieties interact or reactwith the analyte to provide an optical signal, which is indicative ofthe analyte concentration in a body fluid. Preferably, the fluorophorescan be sensed using different platforms, including fluorescenceintensity, lifetime based, anisotropy and ratiometric sensing. Thefluorophores may be used with fluorescence quenchers, enhancers andForster energy-transfer compounds. “Quenchers” are well known in the artand may be any compound that reduces the fluorescence intensity of thefluorophore. “Enhancers” of fluorescence include, but are not limitedto, noble metal surfaces that result in increased fluorescence emission.Compounds useful for energy transfer are any compounds that can absorbthe instant fluorophore's emission and fluoresce at a differentwavelength.

Examples of optical signals include changes in the optical properties,including, but not limited to, a change in color, changes in intensity(absorbance or fluorescence) at the same or different wavelengths, aspectral (absorption or emission) shift, changes in lifetime ofluminescence (fluorescence, phosphorescence, and the like). A change incolor can be observed by the naked eye and can be used in qualitative orsemi-quantitative assays.

A preferred embodiment of the invention includes a fluorescent phenylboronic acid compound, wherein the fluorophore moiety comprises aheterocyclic quaternary nitrogen (a ring nitrogen) linked through aphenyl ring with the boronic acid moiety. Preferably, the fluorescentboronic acid compound is sensitive to the binding of monosaccharides,e.g., glucose and fructose, as well as chloride and iodide.

Measurement of monosaccharide, chloride or iodide concentration can bebased upon measuring any change of fluorescence, described herein.Measurements may be performed with the fluorophore compound free insolution, contained in a matrix or bound to a substrate. Any variationsare also possible, as the fluorescent boronic acid-containing compoundsmay be bound to other compounds in solution, such as to an antibody orprotein. A substrate may be a bead in solution to which the fluorescentcompound is bound. Thus the fluorophores of the present invention may beused in a diagnostic kit for measuring the concentration ofmonosaccharides in bodily fluids.

Additionally, the invention relates to a biocompatible sensor fordetecting/measuring analytes, in particular glucose, in tears and amethod of using said biocompatible sensor. The biocompatible sensor ofthe invention may comprise, consist essentially of, or consist of anophthalmic device including a polymer matrix and the fluorophore inand/or on the polymeric matrix. Preferably, the ophthalmic device is anoff-the-shelf, disposable plastic contact lens.

For the purpose of defining this invention, the most preferred lens is acontact lens, particularly a soft contact lens that may be used on adaily basis or for extended wear. A soft hydrogel lens is the lens mostcommonly worn for extended wear applications, and a poly(vinyl alcohol)(PVA) lens is most commonly worn for disposable daily use.

If the lens is an extended wear type, preferably, the polymer from whichthe lens is derived is formed from polymerizing a monomer from the classof hydroxy esters of acrylic acid or methacrylic acid. The preferredmonomer is hydroxyethylmethacrylate (HEMA). Advantageously, acrosslinking agent is added to the monomer composition from which thepolymeric lens is derived to enhance the mechanical strength of the lensand consequently its handling properties. Crosslinking agents that canbe used are polyfunctional monomers, such as ethylene glycoldimethacrylate (EGDMA).

In the event a daily disposable lens is preferred for a sensing device,a preferred group of lens-forming materials are prepolymers that arewater-soluble and/or meltable. It would be advantageous that alens-forming material comprises primarily one or more prepolymers thatare preferably in a substantially pure form, e.g., purified byultrafiltration. Examples of preferred prepolymers include, but are notlimited to: water-soluble crosslinkable poly(vinyl alcohol) prepolymersas described in U.S. Pat. Nos. 5,583,163 and 6,303,687, which areincorporated by reference herein in their entireties; water-solublevinyl group-terminated polyurethane, which is obtained by reacting anisocyanate-capped polyurethane with an ethylenically unsaturated amine(primary or secondary amine) or an ethylenically unsaturated monohydroxycompound; derivatives of polyvinyl alcohol, polyethyleneimine orpolyvinylamine, which are disclosed in U.S. Pat. No. 5,849,841, which isincorporated by reference herein in its entirety; a water-solublecrosslinkable polyurea prepolymer as described in U.S. Pat. No.6,479,587, which is incorporated by reference herein in its entirety;crosslinkable polyacrylamide; crosslinkable statistical copolymers ofvinyl lactam, MMA and a co-monomer, which are disclosed in EP 655,470and U.S. Pat. No. 5,712,356; crosslinkable copolymers of vinyl lactam,vinyl acetate and vinyl alcohol, which are disclosed in EP 712,867 andU.S. Pat. No. 5,665,840; polyether-polyester copolymers withcrosslinkable side chains which are disclosed in EP 932,635; branchedpolyalkylene glycol-urethane prepolymers disclosed in EP 958,315 andU.S. Pat. No. 6,165,408; polyalkylene glycol-tetra(meth)acrylateprepolymers disclosed in EP 961,941 and U.S. Pat. No. 6,221,303; andcrosslinkable polyallylamine gluconolactone prepolymers disclosed in WO00/031550.

The lens can be lathe cut from a polymeric lens blank, or it can bepolymerized in a mold shaped in the form of a lens, with or without thepresence of an inert diluent. In either case, the hydrogel lens isdesirably swollen in water so that the composition of the lens is atleast 30 weight percent water.

The lens can be impregnated with the fluorophores of the presentinvention using conventional methods. For example, the lens can beimmersed in a solvent which swells the lens and dissolves thefluorophore. The preferred solvents are volatile, short chain alcoholicsolutions, e.g., ethanol. The solution is preferably a dilute aqueoussolution with the concentration of the fluorophore ranging from about0.1 to about 25 weight percent, but preferably around 1 to about 10weight percent. The lens is left in the aqueous solution for a timesufficient for the fluorophore to penetrate and subsequently allow forthe lens to equilibrate. Typically, this period of time can rangebetween 2 to 3 hours. Afterwards, the lens is removed from solution, andthe solvent is removed by simply allowing the lens to dry in air.

Alternatively, the lens can be impregnated by stirring the lens for atleast several minutes in a suspension of molten fluorophore in water orbuffered saline.

It is also possible to coat the surface of the lens with thefluorophore. This may be particularly desirable when the lens is a hardlens or a soft hydrophobic lens. The coating of the outer surfaces ofthese lenses can be accomplished using conventional methods, such as byspraying, dipping or coating with a roller. The resulting coating isoptically clear, resistant to most solvents and to temperature changes,and does not delaminate, flake or crack. The coating typically is aboutten microns or less in thickness, although the thickness of the coatingmay be varied by well-known techniques.

The invention further provides methods for making compositions includingthe fluorophores of the present invention, and applying them to contactlens or similar substrates to form a coating or layer of the fluorophorethereon. The method for making the composition generally comprisesproviding a solution, dispersion or suspension of the fluorophore andapplying the solution to the substrate to form the matrix. The coatingmay be attached to and/or immobilized on the contact lens by anyappropriate method, including covalent bonding, ionic interaction,coulombic interaction, hydrogen bonding, crosslinking (e.g., ascrosslinked (cured) networks) or as interpenetrating networks, forexample. If a crosslinked coating is desired, the fluorophore first iscombined with a crosslinking agent. Typically, both the fluorophore andthe crosslinker will be in liquid form (e.g., in a solution, dispersionor suspension), and the two solutions are combined, forming a liquidmixture. The fluorophore (with or without a crosslinker) can be appliedto the substrate of choice by any suitable means for applying a liquidcoating. If a crosslinker is present, the fluorophore solution may besubjected to crosslinking conditions, that may include thermal curing,ultraviolet curing, chemical curing or other curing methods.

The amount of the fluorophore that is effective as a sensitive sensingagent will depend on numerous factors. However, this amount can bereadily determined empirically. For most instances, and particularlywhen it is desired to determine the level of glucose, the amount offluorophore impregnated in the lens or coated on its surface shouldrange from about 1 to about 10.0 percent of the weight of the lens or inamount sufficient to react with at least the highest concentration ofglucose suspected in the tears of the testing individual.

Advantages of the preferred ophthalmic sensing device, e.g., adisposable contact lens, is the non-invasive nature of the contact lensand the sensing of the change in optical signal, which makes it anattractive method for monitoring physiological conditions, whether inresponse to illness or drug compliance.

An ophthalmic lens according to embodiments of the invention can be usedin an analyte sensor system. The analyte sensor system comprises anophthalmic lens and a detector configured to detect the intensity offluorescence of the novel phenyl boronic acid-doped contact lens in thepresence of analyte. For example, the detector may include afluorophotometer. Construction of such devices is well known in the art.

Light with wavelengths that will excite the fluorescent label can beprovided, for example, by a laser or a light source, such as alight-emitting diode. A fluorophotometer suitable for use withembodiments of the invention can be constructed using a light-emittingdiode from Power Technology, Inc. (Little Rock, Ark.) (see March et al.,Diabetes Technol. & Ther., 2, 27-30 (2000)).

The detector can be a free-standing device, a table-top device, or ahand-held device. For convenience, the detector can be a miniaturizeddevice and may be worn or carried as a personal accessory, for example,mounted in the frame of a pair of eyeglasses, clipped to an article ofclothing, such as a shirt or sweater, hung around the neck, worn aroundthe wrist, or clipped to a belt or a key ring.

If desired, the analyte sensor system also can comprise a transmitterconfigured to transmit a signal representing whether the analyte isdetected and/or an amount of the analyte that is detected. A deviceconfigured to vary the concentration of the analyte in a body fluid ortissue, such as an infusion pump or other pump, may receive the signaland may vary the concentration in response to the signal. The signalfrom the analyte sensor system may comprise a continuous ordiscontinuous telemetry signal generated by the detector. The pump may,in response to the signal, adjust the levels of the analyte in the bodyby providing the user with the appropriate amount of a regulator moiety,such as insulin. Infusion pumps are well known in the art for deliveringa selected medication to a patient including humans and other animals inaccordance with an administration schedule which can be preselected or,in some instances, preprogrammed. Pumps for use in this invention can beworn externally or can be directly implanted into the body of a mammal,including a human, to deliver a specific medication such as insulin tothe mammal in controlled doses over an extended period of time. Suchpumps are well known and are described, for example, in U.S. Pat. Nos.5,957,890, 4,923,375, 4,573,994, and 3,731,681.

It has been determined that the pH of disposable plastic contact lensesis approximately 6.1 and unbufferable, and that the polarity of saidlens approximates that of methanol. As such, this substantially reducesthe dynamic range for sensing.

Because of the necessity of measuring analyte, e.g., glucose, changes influids at or below physiological pH, i.e., pH˜5-8, preferably 6-8, thepK_(a) of the molecular sensing moiety is preferably lowered relative topublished boronic acid fluorophores (BAFs), which typically have apK_(a)>>7 when in the boronate diester, i.e., glucose-bound, form, andtherefore are not sensitive to glucose concentration changes belowphysiological pH.

Towards that end, the pK_(a) of phenyl boronic acid was lowered bychoosing appropriate substituents for the phenyl boronic acid molecule.For example, the addition of an electron withdrawing group andoptionally an electron donating group to the phenyl boronic acidmolecule reduces and increases the pK_(a) of the boronic acid diesterform, respectively. Knowing this, novel phenyl boronic acid derivativeswere synthesized, which have lower glucose-bound pK_(a) values thanpreviously published phenyl boronic acid derivatives, said novel phenylboronic acid compounds being superiorly sensitive to analyte, e.g.,glucose, concentration changes at or below physiological pH.

The novel phenyl boronic acid compounds described herein comprise aquinolinium moiety as a fluorescent indicator and a boronic acid moietyas a chelating group. Referring to FIG. 2, representations of the novelphenyl boronic acid compounds described herein are shown, each having aquinolinium backbone. In the compounds of FIG. 2, X is Cl⁻, Br⁻ or I⁻and R is selected from the group consisting of H, straight chain orbranched C₁-C₄ alkyl group, C₁-C₄ alkoxy group, aryl group, hydroxyl,cyano, sulfonyl, and NR¹R², wherein R¹ and R² may be the same as ordifferent from one another and is independently selected from the groupconsisting of H and C₁-C₄ alkyl groups. Further, although the boronicacid moiety in the compounds of FIG. 2 is shown in the ortho- position,the positioning of the boronic acid group may be meta- or para- relativeto the quinolinium backbone.

Preferred quaternary nitrogen heterocyclic compounds include compound Bof FIG. 2, wherein R is —CH₃, —OCH₃ or —NH₂ and X is Br.

Advantages of the novel phenyl boronic acid compounds described hereininclude, but are not limited to, excellent water solubility, simpleone-step synthesis, long fluorescence lifetimes, charge stabilization,high quantum yields, increased glucose sensitivity, and compatibilitywith existing laser and light emitting diode excitation sources.

The method of using the novel phenyl boronic acid derivatives todetermine blood monosaccharide, chloride or iodide concentrations can beprovided in a kit, together with instructions for measuring the analyteconcentrations. The invention provides kits which are intended forindividual patient use, as well as kits for medical practitioners.

The ophthalmic lens according to the embodiments of the invention canalso be provided in a kit, together with instructions for measuringanalyte concentrations. The invention provides kits which are intendedfor individual patient use, in which the ophthalmic lens typically is acontact lens, as well as kits for medical practitioners, which cancomprise any of the ophthalmic lenses or their equivalents describedherein.

The invention in another aspect encompasses fluorescence compounds thatcontain reactive groups useful for covalent attachment to a polymericmatrix of an ophthalmic device, e.g., contact lens, or other substrate,and are capable of excellent response to glucose.

Such covalently attachable compounds contain a quaternary nitrogenheterocyclic nucleus and boronic acid functionality, as suitable forsensing glucose in the ophthalmic device or other substrate. Thefluorescent nucleus of such compounds has a sugar bound pKa that iscompatible with mildly acidic environments, such as are characteristicof contact lenses. The compounds are effective to transduce glucoselevels in contact lenses, which have a polarity similar to methanol.

The covalently attachable fluorescence compounds respond to glucose atlevels that are well over the physiological tear level. The reactivegroups of such compounds enable the compounds to be covalently bonded toa contact lens, but they do not alter or influence the signaling ofglucose. These reactive groups can be of any suitable type, as effectiveto form covalent bonds with the substrate, e.g., contact lens, material.

It will be appreciated that the fluorescence compounds should have astructure that avoids steric hindrance upon glucose binding, since theresponse is deteriorated by such steric hindrance.

In one specific embodiment, the reactive groups imparting covalentbonding function to the compound are allyl groups. In other embodiments,the reactive functionality imparting covalent bonding character to thefluorescence compound can be constituted by other ethylenicallyunsaturated groups or moieties, e.g., ethenyl or other pendant alkenylfunctionality, acryloxy functionality, etc., as may be appropriate inview of the specific composition and character of the substrate to whichthe fluorescence compound is to be covalently bonded. In general, thereactive functionality can be any functionality that is reactive withthe substrate to provide a covalently bound analyte-detectingcomposition that in the presence of the analyte produces an opticalsignal indicative of analyte level in the physiological fluid or othermedium being monitored for the presence or concentration of the analyte.

By way of example, compounds covalently bondable to ophthalmic device,e.g., contact lens, substrates, can include one or more of the followingcompounds (L)-(N):

Compounds of such type are readily synthesized, within the skill of theart and without undue experimentation, to provide heterocyclicquaternary nitrogen molecules containing at least one heterocyclicquaternary nitrogen (a ring nitrogen) that is linked through a phenylring with a boronic acid group —B(OH)₂, e.g., with a benzyl grouppendant from the heterocyclic quaternary nitrogen and having a boronicacid substituent on the phenyl ring of the benzyl group. Suitablecompounds can include multiple heterocyclic quaternary nitrogens eachhaving a benzyl moiety bonded thereto, with boronic acid substituents onone or more of the phenyl rings of such benzyl moieties. For example, inreference to the above illustrative compounds (L)-(N), the compounds caninclude two such benzyl moieties, with boronic acid substituents —B(OH)₂on one or both phenyl rings of the respective benzyl moieties, inrelation to the corresponding unboronated compound (O):

It will therefore be appreciated that the invention provides variousclasses of compounds that are effective for sensing of analytes insolution, e.g., blood, serum, urine, water, etc. Such compounds can beutilized as unanchored sensing molecules in the solution or otherenvironments. Alternatively, such compounds can be immobilized on asubstrate or be incorporated in support media for a wide variety ofsensing applications.

Glucose-sensing contact lenses form a preferred embodiment of theinvention, and are usefully employed by diabetics for monitoring insulinin a simple and non-invasive manner. In addition, monitoring of otherphysiological analytes in a contact lens having fluorophore compoundsassociatively incorporated in the polymeric matrix of the contact lens,or covalently bonded to such matrix, include, by way of example, andwithout limitation: monitoring lithium for patient stability and drugcompliance; determining cholesterol levels in connection with treatmentby cholesterol-lowering therapeutic agents; monitoring sodium andpotassium levels for hypertension treatment; determining exposure tobiological agents by tear analysis of military or first responderpersonnel in areas suspected or susceptible to incursion of adversebiological agents; sensing of cyanide by tear analysis of workers in theplastics industries who may encounter cyanide as a work-related toxin;and monitoring drug compliance in large clinical screens.

Various sensing applications within the scope of the present inventionmay utilize compounds that are highly specific in sensitivity to atarget analyte species, as single compound uses that areanalyte-specific in character. The invention also contemplates the useof single compounds that are sensitive to a wide variety of analytes andconditions, e.g., two or more of glucose, fructose, sodium, potassium,lithium, histamine, cholesterol, cyanide, fluoride, pH and otherenvironmental conditions. Further, the invention contemplates theprovision of multicomponent mixtures of sensing compounds, providing thecapability for sensing of a spectrum of analyte target species.

The features and advantages of the invention are more fully shown withrespect to the following illustrative examples and embodiments.

Methods and Materials

D-Glucose and D-fructose were purchased from Sigma and used as received.All solvents used were HPLC grade and purchased from Aldrich.1. Preparation of o-, m- and p-N-(boronobenzyl)-6-methylquinoliniumbromide (BMQBA) and N-benzyl-6-methylquinolinium bromide (BMQ)

The boronic acid containing fluorescent molecular sensing moieties o-,m- and p-BMQBA and the control compound MBQ were prepared using thefollowing generic one step synthetic procedure, described herein forMBQ. Equimolar amounts of 6-methylquinoline and benzylbromide weredissolved in 10 mL dry acetonitrile in a 25 mL round bottomed flaskequipped with a magnetic stirrer. The reaction mixture was allowed tostir under an inert atmosphere for 24 hrs at room temperature. Duringthis time, a quantitative amount of quaternized salt was precipitated asa colorless solid. The solid product recovered by filtration was washedseveral times with dry acetonitrile and then dried under vacuum for 12hrs. BMQ ¹H NMR (D₂O) δ (ppm): 2.5 (s, 3H); 6.2 (s, 2H); 7.2-7.5 (m,5H), 7.8 (d, 1H); 8.0 (m, 2H); 8.15 (d, 1H); 9.0 (d, 1H); and 9.3 (d,1H). HRMS (FAB+, H₂O) m/e calculated: 234.1283 (M⁺-Br), found: 234.1291(M⁺-Br).

The corresponding o-, m- and p-boronobenzyl bromides are employedinstead of benzyl bromide to obtain the isomeric boronic acidderivatives o-, m- and p-BMQBA, respectively. o-BMQBA ¹H NMR (D₂O) δ(ppm): 2.7 (s, 3H); 6.5 (s, 2H); 7.1 (s, 1H), 7.4-7.5 (m, 2H); 8.0-8.3(m, 4H); 8.5 (d, 1H); 8.95 (d, 1H); and 9.2 (d, 1H). HRMS (FAB+, H₂O)m/e calculated: 346.1978 (M⁺-Br), found: 346.1960 (M⁺-Br). m-BMQBA ¹HNMR (D₂O) δ (ppm): 2.5 (s, 3H); 6.2 (s, 2H); 7.3-7.5 (m, 2H), 7.6 (s,1H); 7.7 (d, 1H); 7.9 (d, 1H); 8.0 (m, 2H); 8.2 (d, 1H); 9.0 (d, 1H) and9.25 (d, 1H). HRMS (FAB+, H₂O) m/e calculated: 346.1978 (M⁺-Br), found:346.1988 (M⁺-Br). p-BMQBA ¹H NMR (D₂O) δ (ppm): 2.55 (s, 3H); 6.2 (s,2H); 7.25 (d, 2H), 7.7 (d, 2H); 7.9 (t, 1H); 8.0-8.2 (m, 3H); 9.0 (d,1H); and 9.25 (d, 1H). HRMS (FAB+, H₂O) m/e calculated: 346.1978(M⁺-Br), found: 346.1960 (M⁺-Br).2. Preparation of o-, m- and p-N-(boronobenzyl)-6-methoxyquinoliniumbromide (BMOQBA) and N-benzyl-6-methoxyquinolinium bromide (BMOQ)

The control compound BMOQ was conveniently prepared using the genericone-step procedure described above for the synthesis of BMQ, wherein6-methoxyquinoline was used instead of 6-methylquinoline. BMOQ ¹H NMR(CD₃OD) δ (ppm): 4.1 (s, 3H); 6.3 (s, 2H); 7.3-7.5 (m, 5H); 7.85 (m,2H); 8.15 (t, 1H); 8.45 (d, 1H); 9.2 (d, 1H) and 9.4 (d, 1H). HRMS(FAB+, H₂O) m/e calculated: 250.1232 (M⁺-Br), found: 250.1222 (M⁺-Br).

The corresponding o-, m- and p-boronobenzyl bromides are employedinstead of benzyl bromide to obtain the isomeric boronic acidderivatives o-, m- and p-BMOQBA, respectively. o-BMOQBA ¹H NMR (CD₃OD) δ(ppm): 4.05 (s, 3H); 6.5 (s, 2H); 7.1 (s, 1H); 7.3-7.5 (m, 2H); 7.8-8.0(m, 4H); 8.5 (t, 1H); 8.8 (d, 1H) and 9.1 (d, 1H). HRMS (FAB+, H₂O) m/ecalculated: 362.1927 (M⁺-Br), found: 362.1960 (M⁺-Br). m-BMOQBA ¹H NMR(CD₃OD) δ (ppm): 4.0 (s, 3H); 6.2 (s, 2H); 7.35-7.55 (m, 2H); 7.6-7.8(m, 4H); 8.0 (t, 1H); 8.25 (d, 1H); 8.95 (d, 1H) and 9.15 (d, 1H). HRMS(FAB+, D₂O) m/e calculated: 362.1927 (M⁺-Br), found: 362.1848 (M⁺-Br).p-BMOQBA ¹H NMR (CD₃OD) δ (ppm): 4.0 (s, 3H); 6.2 (s, 2H); 7.25 (d, 2H),7.5-7.8 (m, 4H); 8.0 (t, 1H); 8.2 (d, 1H); 8.95 (d, 1H) and 9.15 (d,1H). HRMS (FAB+, H₂O) m/e calculated: 362.1927 (M⁺-Br), found: 362.1956(M⁺-Br).

3. Absorption and Emission Studies of BMQBA in the Presence and Absenceof Monosaccharides

The measurement of monosaccharides can be based upon measuring any ofthe changes in fluorescence of the disclosed fluorescent compounds, asreadily determined by one skilled in the art. Measurements may beperformed with the fluorophore free in solution, contained in a matrixor bound to a substrate or other compounds in solution, e.g., antibodiesor proteins.

All steady state fluorescence measurements were performed in a 4×1×1 cmfluorometric plastic cuvette, using a Varian Cary Eclipse fluorometer,and all absorption measurements were performed using a Varian UV/VIS 50spectrophotometer.

Stability (K_(S) (mM⁻¹) and dissociation (K_(D)) constants were obtainedby fitting the titration curves with sugar to the relation:$I = \frac{I_{\min} + {I_{\max}K_{S{\lbrack{sugar}\rbrack}}}}{1 + K_{S{\lbrack{sugar}\rbrack}}}$where I_(min) and I_(max) are initial (no sugar) and final (plateau)fluorescence intensities of the titration curves and K_(D)=(1/K_(S)).

Time-solved intensity decays were measured using reverse start-stoptime-correlated single-photon timing (TCSPC) with a Becker and HicklGmbh 630 SPC PC card and unamplified MCP-PMT. Vertically polarizedexcitation at ˜372 nm was obtained using a pulsed LED source (1 MHzrepetition rate) and a dichroic sheet polarizer. The instrumentalresponse function was ˜1.1 ns fwhm. The emission was collected at themagic angle (54.7°) using a long pass filter (Edmund Scientific) whichcut-off the excitation wavelengths.

The intensity decays were analyzed in terms of the multi-exponentialmodel:${I(t)} = {\sum\limits_{i}{\alpha_{i}{\exp\left( {{- t}/\tau_{i}} \right)}}}$where α_(i) are the amplitudes and τ_(i) are the decay times,Σα_(i)=1.0. The fractional contribution of each component to thesteady-state intensity is given by:$f_{i} = \frac{\alpha_{i}\tau_{i}}{\sum\limits_{i}{\alpha_{i}\tau_{i}}}$

The mean lifetime of the excited state is given by:$\overset{\_}{\tau} = {\sum\limits_{i}{f_{i}\tau_{i}}}$

The values of α_(i) and τ_(i) were determined by non-linear leastsquares impulse reconvolution with a goodness-of-fit χ² _(R) criterion.

A representative absorption and emission spectra for o-BMQBA in water isshown in FIG. 3B, which corresponds to all three isomers and BMQ. BMQBAhas a strong absorption band at ˜320 nm, which can be assigned to n→π*transitions, and an excitation band at ˜427 nm, and as such, has a largeStokes shift of about 100 nm, which is ideal for fluorescence sensing.The quantum yield values of the BMQBA compounds in water relative toN-(3-sulfopropyl)-6-methoxyquinolinium (SPQ) (φ_(f)=0.53 in water) areshown in Table 1. The low quantum yields and relatively short lifetimeof the BMQBA and BMQ molecules can be attributed to a photo-inducedelectron transfer, whereby the phenyl ring acts as the donor and thequinolinium moiety acts as the acceptor. TABLE 1 Spectral properties inwater, pK_(a) values in the presence and absence of 100 mM sugar, anddissociation constants of the molecular sensing moieties in pH 7.5phosphate buffer with glucose and fructose. o-BMQBA m-BMQBA p-BMQBA BMQλ_(abs) (max)/nm 319 322 322 322 λ_(em) (max)/nm 427 427 427 427 φ_(f)0.043 0.025 0.023 0.045 τ_(f)/ns (mean lifetime) 4.01 3.72 2.10 2.59pK_(a) (buffer) 6.92 7.75 7.80 — pK_(a) 6.18 6.85 6.95 — (buffer +glucose) pK_(a) 5.08 5.05 5.45 — (buffer + fructose) K_(d)/mM (glucose)100 479 370 — K_(d)/mM (fructose) 4.7 13.2 13.8 —

The emission spectra of o-BMQBA in the presence of varyingconcentrations of fructose and glucose are shown in FIG. 4A and FIG. 4B,respectively. It can be seen that the fluorescence intensity of o-BMQBAin pH 7.5 phosphate buffer is inversely proportional to the glucose andfructose concentrations. Both m-BMQBA and p-BMQBA showed similarresponses towards glucose, fructose and other monosaccharides. A plot ofthe intensity ratio, I′/I, at λ=427 nm, where I′ and I represent thefluorescence intensities in the absence and presence of sugar,respectively, is shown in FIG. 4C. As expected, BMQBA shows a higheraffinity for fructose than for glucose (James, T. D., et al., Agnew.Chem. Int. Ed. Engl., 33, 2207 (1994); James, T. D., et al., J. Am.Chem. Soc., 117, 8982 (1995); Bielecki, M., et al., J. Chem. Soc. PerkinTrans., 2, 449 (1999); Dicesare, N., et al., Anal. Biochem., 294,154-160 (2001); Dicesare, N., et al., J. Photochem. Photobiol. A, 143,39-47 (2001); Dicesare, N., et al., Org. Lett., 3(24), 3891-3893 (2001);Dicesare, N., et al., Tetrahedron Lett., 43, 2615-2618 (2002); Dicesare,N., et al., J. Phys. Chem. A, 105, 6834-6840 (2001)). It is noted thatthe concentration of fructose in blood is ˜10 times lower than glucose,a relationship which is also thought to occur in tears (N. Dicesare, et.al., J. Bio. Med. Opt., 7(4), 538-545 (2002)). Hence, fructose is notthought to be a major interferent in physiological fluids.

The emission spectra of o-BMQBA at varying pH values in the absence ofsugar (FIG. 5C) and in the presence of 100 mM fructose and 100 mMglucose (FIGS. 5A and 5B, respectively), show that the fluorescenceintensity of o-BMQBA decreases with increasing pH (3→11), both with andwithout sugar. Notably, BMQ shows no change in intensity with pH change.A normalized plot of intensity at 427 nm as a function of pH is shown inFIG. 6, wherein I is the emission intensity at the specified pH andI_(o) is the initial emission intensity at pH 3.0. Though not to bebound by theory, it is likely that the change in fluorescence intensityrelative to pH value is attributed to the change in hybridization of theboron atom with increasing pH. At low pH values, the boronic acid groupis an electron-deficient Lewis acid that is sp²-hybridized and thus istrigonal planar. As the pH increases, the anionic form of the boronicacid begins to form, which corresponds to a more electron richsp³-hybridized boron atom and an octahedral shape. The change in thegeometry of the boron atom induces the fluorescence spectral changes ofthe molecular sensing moieties.

The pK_(a) values obtained from the normalized intensity plot of FIG. 6are presented in Table 1. Based on previously reported pK_(a) values forboronic acid compounds, these pK_(a) values are the lowest reported fora phenyl boronic acid derivative. The quaternary nitrogen of thequinolinium moiety not only reduces the pK_(a) of the compound, but alsostabilizes the anionic boronate diester complex (represented by D inFIG. 1), which may explain the affinity of sugar for these novelcompounds. Importantly, the large decrease in the pK_(a) of the boronicacid-sugar complex relative to the uncomplexed boronic acid, allows forthe quantitative detection of monosaccharides at or below physiologicalpH because substantial optical changes are observed.

The emission spectra of o-BMQBA when both the pH media and glucoseconcentrations are varied are shown in FIG. 7, wherein FIGS. 7A, 7B, 7Cand 7D correspond to pH 5, 6, 7, and 8 buffers, respectively. It can beseen that as the pH is increased from 5 to 8 with an increasingconcentration of glucose, there is a quantifiable reduction influorescence intensity. This is again attributed to the increasedelectron density on the boron atom as the pH increases, which results inthe partial neutralization of the positively charged quaternary nitrogenof the quinolinium moiety, which has been termed herein “chargeneutralization-stabilization mechanism.” Just as an increased pHincreases the electron density on the boron atom, the binding of glucoseat the boron atom also induces charge neutralization-stabilization, asschematically shown below. With stabilization, e.g., binding of glucoseto the boronic acid moiety, there is a quantifiable reduction influorescence intensity.

The intensity ratio for o-BMQBA in buffered media (pH 5-8) at variousglucose concentrations is shown in FIG. 8A, wherein I′ corresponds tothe intensity in the absence of glucose and I corresponds to theintensity at the specified glucose concentration. In addition, theintensity ratio for o-BMQBA in buffered media (pH 5-8) at low glucoseconcentrations, typical of those found in tears, is shown in FIG. 8B.Correspondingly, the intensity ratio for o-BMQBA in buffered media (pH5-8) at various fructose concentrations is shown in FIGS. 9A and 9B.

For glucose, in the pH 6 to 7 range, it can be seen that for 60 mMglucose, there is a 1.4→2.1 fold decrease in fluorescence intensity (seeFIG. 8A) and about a 10% decrease in fluorescence intensity at 0.60 mMglucose (see FIG. 8B). The latter change is especially important whenassessing the concentration of glucose in tears because tear glucoselevels are reported to change from ˜500 μM to 5 mM for diabetics(Gasser, A. R., et al., Am. J. Ophthalmology, 65(3), 414-420 (1968);Das, B. N., et al., J. Indian Med. Assoc., 93(4), 127-128 (1995)). In acorresponding manner, for fructose in the pH 6 to 7 range, there is a4→6 fold decrease in fluorescence intensity at 50 mM fructose and a1.3→2.0 fold decrease in intensity at 0.60 mM fructose. These aremeasurable differences which allow for the quantification ofmonosaccharide concentrations.

Because physiological fluids contain chloride ions, which are well knownquenchers of quinolinium fluorescence (Geddes, C. D., Meas. Sci.Technol., 12(9), R53-R88 (2001); Geddes, C. D., et al., J. HeterocyclicChem., 36(4), 949-951 (1999); Geddes, C. D., et al., Anal. Biochem.,293(1), 60-66 (2001)), the fluorescence intensity and intensity ratiosof o-BMQBA in pH 7.5 buffer having 100 mM NaCl with varying quantitiesof fructose or glucose was measured and shown in FIGS. 10 and 11,respectively, where I′ corresponds to the intensity in the absence offructose and I corresponds to the intensity at the specified fructoseconcentration. It can be seen that although chloride quenched theintensity of the emission in the presence of fructose, the intensityratio change for fructose is still significant, amounting to a 4-folddecrease in fluorescence intensity at 60 mM fructose (see FIG. 10B). Thequenching effect was less significant at lower concentrations offructose (see FIG. 10C) and for glucose concentrations, both high andlow (see FIGS. 11B and 11C). This minimal quenching effect for BMQBA issupported by the calculated Stern-Volmer constants, K_(SV), for theBMQBA compounds in water, which were determined to be 44.0 M³¹ ¹, 20.0M⁻¹, 17.0 M⁻¹ and 35.0 M⁻¹ for o-BMQBA, m-BMQBA, p-BMQBA and BMQ,respectively. These are small quenching constants that are readilyaccounted for using simple corrections in the fluorescence signal, asreadily determined by one skilled in the art of fluorescence.

Notably, because chloride ions are known in the art to quench thefluorescence intensity of quinolinium derivatized compounds, theheterocyclic compounds can be used to qualitatively and quantitativelydetermine the presence of chloride ions in a solution. Moreover, becausethe quenching of fluorescence is not a selective process, anyfluorophore quenched by chloride is also quenched by bromide and iodideto an even more substantial extent, allowing for the determination ofbromide or iodide concentrations as well. Generally, fluorophores thatare quenched by chloride are not quenched by fluoride, which is oftenattributed to the “heavy-atom effect” (Geddes, C. D., Meas. Sci.Technol., 12, R53 (2001); Lakowicz, J. R., Principles of FluorescenceSpectroscopy, 2^(nd) ed., Kluwer/Academic Plenum Publishers, New York,1997). It is however noted that the inventors have surprisinglydiscovered that N-(2-boronobenzyl)-6-aminoquinolinium bromide (BAQBA),which has the general structure of B in FIG. 2, is very sensitive tofluoride and is able to detect fluoride concentrations below about 50mM.

4. Absorption and Emission Studies of BMOQBA in the Presence and Absenceof Monosaccharides

As introduced above, the measurement of monosaccharides can be basedupon measuring any of the changes in fluorescence of the disclosedfluorescent compounds, as readily determined by one skilled in the art.Measurements may be performed with the fluorophore free in solution,contained in a matrix or bound to a substrate or other compounds insolution, e.g., antibodies or proteins.

A representative absorption and emission spectra for o-BMOQBA in wateris shown in FIG. 3A, which corresponds to all three isomers and BMOQ.BMOQBA has a strong absorption band at ˜345 nm, which can be assigned ton→π* transitions, and an excitation band at ˜450 nm, and as such, has alarge Stokes shift of about 100 nm, which is ideal for fluorescencesensing. The quantum yield values of the BMOQBA compounds in waterrelative to N-(3-sulfopropyl)-6-methoxyquinolinium (SPQ) (φ_(f)=0.53 inwater) are shown in Table 2. The lower quantum yields of the BMOQBAmolecules relative to BMOQ can possibly be explained by the interactionbetween the boronate diester (represented by D in FIG. 1) and thepositively charged nitrogen center at neutral pH, which is expected tobe most prominent for the o-BMOQBA isomer, hence the lowest quantumyield. We can only speculate as to the quantum yield differences betweenthe m-BMOQBA and p-BMOQBA compounds, which may be attributed tothrough-bond and through-space mechanisms. TABLE 2 Spectral propertiesin water, pK_(a) values in the presence and absence of 100 mM sugar, anddissociation constants of the molecular sensing moieties in pH 7.5phosphate buffer with glucose and fructose. o-BMOQBA m-BMOQBA p-BMOQBABMOQ λ_(abs) (max)/nm 318, 346 318, 347 318, 346 318, 347 λ_(em)(max)/nm 450 450 451 453 φ_(f) 0.46 0.51 0.49 0.54 τ_(f)/ns 26.7 25.924.9 27.3 (mean lifetime) pK_(a) (buffer) 7.90 7.70 7.90 — pK_(a)(buffer + 6.62 6.90 6.90 — glucose) pK_(a) (buffer + 4.80 5.00 5.45 —fructose) K_(d)/mM 49.5 1000 430 — (glucose) K_(d)/mM 0.65 1.8 9.1 —(fructose)

The emission spectra of o-BMOQBA in the presence of varyingconcentrations of glucose and fructose are shown in FIG. 12A and FIG.12B, respectively. It can be seen that the fluorescence intensity ofo-BMOQBA in pH 7.5 phosphate buffer is inversely proportional to theglucose and fructose concentrations. Both m-BMOQBA and p-BMOQBA showedsimilar responses towards glucose, fructose and other monosaccharides. Aplot of the intensity ratio, I′/I, at λ=450 nm, where I′ and I representthe fluorescence intensities in the absence and presence of sugar,respectively, is shown in FIG. 12C. As expected, BMOQBA shows a higheraffinity for fructose than for glucose (James, T. D., et al., Agnew.Chem. Int. Ed. Engl., 33, 2207 (1994); James, T. D., et al., J. Am.Chem. Soc., 117, 8982 (1995); Bielecki, M., et al., J. Chem. Soc. PerkinTrans., 2, 449 (1999); Dicesare, N., et al., Anal. Biochem., 294,154-160 (2001); Dicesare, N., et al., J. Photochem. Photobiol. A, 143,39-47 (2001); Dicesare, N., et al., Org. Lett., 3(24), 3891-3893 (2001);Dicesare, N., et al., Tetrahedron Lett., 43, 2615-2618 (2002); Dicesare,N., et al., J. Phys. Chem. A, 105, 6834-6840 (2001)). Referring to FIG.12C, there is a 2.8-fold change in intensity by the addition of 60 mMsugar. Interestingly, useful intensity changes are shown in the 2 mM→40mM range, which corresponds to the glucose concentration of a healthyperson to the upper limit experienced by a diabetic (DiCesare, N., etal., J. Bio. Med. Opt., 7(4), 538-545 (2002)).

The emission spectra of o-BMOQBA at varying pH values in the absence ofsugar (FIG. 13A) and in the presence of 100 mM glucose and 100 mMfructose (FIGS. 13B and 13C, respectively), show that the fluorescenceintensity of o-BMOQBA decreases with increasing pH (3→11), both with andwithout sugar. Notably, BMOQ shows no corresponding change in intensitywith pH change. A normalized plot of intensity at 450 nm as a functionof pH is shown in FIG. 14, wherein normalized intensity is the emissionintensity at the specified pH, I, relative to the initial emissionintensity at pH 3.0, I_(o). As discussed with reference to o-BMQBA, thechange in the geometry of the boron atom may explain the fluorescencespectral changes of the molecular sensing moieties.

The pK_(a) values obtained from the normalized intensity plot of FIG. 14are presented in Table 2. Comparing the pK_(a) values of previouslyreported boronic acid compounds relative to the novel BMOQBA isomers,these pK_(a) values are relatively low. As discussed with reference too-BMQBA, the lower pK_(a) values can be attributed to the increasedLewis acidity of the boronate diester complex (represented by D in FIG.1). Importantly, the large decrease in the pK_(a) of the boronicacid-sugar complex relative to the uncomplexed boronic acid, allows forthe quantitative detection of monosaccharides at or below physiologicalpH because substantial optical changes are observed.

The emission spectra of o-BMOQBA when both the pH media and glucoseconcentrations are varied are shown in FIG. 15, wherein FIGS. 15A, 15B,15C and 15D correspond to pH 5, 6, 7, and 8 buffers, respectively. Asdiscussed with reference to o-BMQBA, decreasing intensity withincreasing pH is attributed to the “charge neutralization-stabilizationmechanism.” With stabilization, e.g., binding of glucose to the boronicacid moiety, there is a quantifiable reduction in the fluorescenceintensity.

The intensity ratio at λ=450 nm for o-BMOQBA in buffered media (pH 5-8)at various glucose concentrations is shown in FIG. 16A, wherein I′corresponds to the intensity in the absence of glucose and I correspondsto the intensity at the specified glucose concentration. In addition,the intensity ratio for o-BMOQBA in buffered media (pH 5-8) at lowglucose concentrations, typical of those found in tears, is shown inFIG. 16B. Correspondingly, the intensity ratio for o-BMOQBA in bufferedmedia (pH 5-8) at various fructose concentrations is shown in FIGS. 17Aand 17B.

It can be seen that for 60 mM glucose, in the pH 6 to 7 range, there isa 1.3→2.5 fold decrease in fluorescence intensity (see FIG. 16A) andabout a 5-10% decrease in fluorescence intensity at 0.60 mM glucose (seeFIG. 16B). In a corresponding manner, for fructose in the pH 6 to 7range, there is a 2.7→3.7 fold decrease in fluorescence intensity at 60mM fructose and a 10-40% decrease in intensity at 0.50 mM fructose.These are measurable differences that allow for the quantification ofmonosaccharide concentrations.

As presented with reference to o-BMQBA, the quenching of thefluorescence intensity of o-BMOQBA by chloride was tested. Thefluorescence intensity and intensity ratios of o-BMOQBA in pH 7.5 bufferhaving 100 mM NaCl with fructose and glucose was measured and shown inFIGS. 18 and 19, respectively, where I′ corresponds to the intensity inthe absence of fructose and I corresponds to the intensity at thespecified fructose concentration. With regards to glucose, the presenceof chloride quenched the intensity of the emission, as expected(Jayaraman, S., et al., Biophys. Chem., 85, 49-57 (2000)). Thecalculated Stern-Volmer constants, K_(SV), for the BMOQBA compounds inwater were determined to be 170 M⁻¹, 182 M⁻¹, 177 M⁻¹ and 222 M⁻¹ foro-BMOQBA, m-BMOQBA, p-BMOQBA and BMOQ, respectively, which is indicativeof modest quenching of the BMOQBA molecules by the chloride ion.

Importantly, monosaccharide levels are still determinable over the highbackground chloride levels present in the blood and other physiologicalfluids. Moreover, the control compounds, e.g., BMQ and BMOQ, which donot bind monosaccharides because of the lack of a boronic acid moiety,are equally sensitive to chloride ions. As such, it is envisioned thatan alternative embodiment of this invention utilizes both the novelphenyl boronic acid probes and the novel control compounds, to monitormonosaccharide and chloride levels, respectively, simultaneously.

Notably, the BMOQBA molecular sensing moieties were found to havemonoexponential lifetimes (˜24.9 ns→26.7 ns), as compared to the BMQBAmolecular sensing moieties which were biexponential in water (2.10ns→4.01 ns). Notably, the lifetimes of BMOQ and BMQ are 27.3 ns and 2.59ns, respectively. These lifetimes directly correlate with the quantumyields for the respective novel compounds. As previously discussed withregards to the lower quantum yield, the lower lifetime of the BMQBAmolecular sensing moiety may be attributed to a photo-induced electrontransfer mechanism, wherein the phenyl ring of the BMQBA acts as anelectron donor and the quaternary nitrogen heterocyclic center as anelectron acceptor. In addition, the B(OH)₃ ⁻ (represented by B inFIG. 1) present at or above neutral pH further reduces the lifetime ofthe boronic acid molecular sensing moieties (see FIG. 20, where thelifetime of o-BMOQBA decreases with increasing pH).

5. Absorption and Emission Studies of Contact Lenses Containing BMQBA inthe Absence and Presence of Monosaccharides

The ophthalmic sensor device of the invention comprises a contact lensmade from any known suitable lens-forming materials. For example, alens-forming material can be a prepolymer, a mixture of prepolymers, amixture of monomers, or a mixture of one or more prepolymers and one ormore monomers and/or macromers. A lens-forming material can furtherinclude other components, such as a photoinitiator, a visibility tintingagent, UV-blocking agent, photosensitizers, and the like. It should beunderstood that any silicone-containing prepolymers or any silicone-freeprepolymers can be used in the present invention.

A contact lens of the invention can be used for non-invasive monitoringof glucose levels in tears. Glucose levels in tears can be convertedinto blood glucose levels based on correlations between tear glucoselevels and blood glucose levels (Süllmann, Handbuch der PhysiologischenChemie, Vol. II/a, p. 867, Springer, Berlin, 1956; Graymore, The Eye,Vol. I, p. 348, Davson, ed., Academic Press, NY, 1962; De Berardinis etal, Exp. Eye Res., 4, 179 (1965); Pohjola, Acta Ophthalmologica Suppl.,88, (1966); Reim et al., Ophthalmologica, 154, 39-50 (1967); Kinsey &Reddy, in Prince, ed., The Rabbit and Eye Research, C. C. Thomas,Springfield, Ill., 1964, p. 218).

The ophthalmic sensor device of the present invention can be animplantable ophthalmic device. Moreover, because the glucose levels intears may be substantially lower than blood glucose levels, using animplantable sensor device, one can monitor periodically or on demandglucose levels in aqueous humor or interstitial fluid where glucoselevels can be much higher than the glucose levels in tears.

In the present case, daily-disposable contact lenses were used, suppliedby CIBA Vision (Atlanta, Ga., USA), which were stirred in 500 mL waterat 20° C. for 24 hours before post-doping. The contact lenses were a PVAtype photo cured polymer which swells slightly in water. The hydrophiliccharacter of the lens allows for the diffusion of the aqueous analytesin tears. The lenses were subsequently doped by incubating the lenses ina high concentration of a phenyl boronic acid derivative, e.g., BMQBA,solution for 24 hours before being rinsed with Millipore water. Thelenses were used immediately after being doped.

The absorption and emission of the phenyl boronic acid derivative fromthe contact lens were measured using a quartz lens holder. The quartzlens holder has the dimensions 4×2.5×0.8 cm, all four sides being ofoptical quality. The contact lens were mounted onto a stainless steelmount of dimensions 4×2×0.4 cm, which fits tightly into the quartz lensholder. A circular hole in the center of the mount with a 2.5 cm innerdiameter, has a raised quartz lip which enables the lens to be mountedthereon. The mount and holder readily allow for ˜1.5 cm³ of solution tobe in contact with the front and back sides of the lens for the sugarsensing experiments. Excitation and emission were measured using aVarian fluorometer with the concave edge of the lens facing towards theexcitation source, to reduce scattering of the excitation light. It isnoted that measurements performed when the convex edge of the lens facesthe excitation source yielded identical results.

Fluorophore leaching experiments were performed at 20° C. using the lensholder, which contains approximately 1.5 cm³ buffer. A Varianfluorometer was used to measure the intensity change as a function oftime to determine the percent signal change that corresponds toleaching. It is noted that in the absence of a sample, no intensityfluctuations or drifts were observed, indicating stability of thefluorometer Xe-arc source.

FIG. 21A shows the leaching of o-BMQBA from a BMQBA-doped contact lensinto pH 7.5 buffer with time. It is noted that due to the very lowconcentration of fluorophores sensing compounds within the lenses, theamount of unleached fluorophores sensing compounds could not be readilydetermined. As such, the percent loss of fluorescence intensity from thelens as a function of time was determined (see FIG. 21B). This wasperformed simply to provide an indication of how long the lenses shouldbe leached before use. Based on FIG. 21B, the lenses were allowed toleach excess molecular sensing compound for 60 minutes before absorptionand emission measurements or the addition of sugars to the solutions.

Following leaching, buffered solutions of sugars were added to the lensholder. Because the 90% response time, which is the amount of timeneeded for the fluorescence signal to change by 90% of the initialvalue, was approximately 10 minutes, fluorescence spectra were typicallytaken approximately 15 minutes after each sugar addition to allow thelens to reach equilibrium.

It is noted that the shelf-life of the doped contact lens was tested,and it was determined that lenses that had been doped, leached andstored for several months, both wet and dry, gave identical sugarsensing results, indicating no fluorophore-polymer interactions orfluorophore degradation over this time period.

A representative absorption and emission spectra for a o-BMQBA-dopedcontact lens in pH 7.5 buffer is shown in FIG. 22, which corresponds toall three isomers and BMQ. BMQBA has a strong absorption band at ˜320 nmand an excitation band at ˜420 nm, therefore having a large Stokes shiftof about 100 nm.

FIGS. 23A and 24A show the fluorescence intensity of o-BMQBA-dopedcontact lenses with increasing concentrations of fructose and glucose,respectively, when the respective monosaccharides are injected into the1.5 cm³ contact lens volume. Similar to the solution based measurements(see FIGS. 4A and 4B), the molecular sensing moieties show a decrease influorescence intensity with increasing fructose and glucoseconcentration, which was attributed to the complexation of diols withboronic acid and subsequent charge neutralization. Intensity ratioplots, where I′ is the intensity in the absence of fructose and I is theintensity at the specified fructose or glucose concentration, are shownin FIGS. 23B, 23C or 24B, 24C, respectively. As was observed with thesolution measurements, phenyl boronic acid derivatives seem to have agreater affinity for fructose, which is based on the greater response offructose. Notably, however, when the concentration of sugar is <2 mM,the response to both sugars was comparable (Badugu, R., et al., J.Fluorescence, 13, 371-374 (2003)).

Importantly, the doped contact lens is reversibly responsive to theaqueous monosaccharides at physiological tear concentrations, thusallowing the continuous monitoring of tear analytes.

FIG. 25 shows the differences in response of the o-BMQBA-doped contactlenses towards the sugars relative to the solution-based studies.Interestingly, the response in the lens was greater than in thesolutions when fructose was added to the lens holder (see FIGS. 25A and25B), while just the opposite was observed for high concentrations ofglucose (FIGS. 25C and 25D). It is speculated that this may be due togreater leaching of the glucose-bound form of the phenyl boronic acidcompounds from the contact lens and/or their displaced solubility withinthe contact lens polymer. Importantly, at low concentrations of glucose,the response in the lens was nearly equivalent to that previouslyobserved with the solution-based studies, amounting to a 5-10% change inintensity at 0.50 mM glucose.

Most boronic acid probes suffer from too low a binding constant to beable to detect and determine tear glucose concentrations, thus makingthem useless for sensing applications. However, the novel fluorophoresdisclosed herein have higher binding constants and as such, are able todetermine tear glucose concentrations and are unperturbed at lowconcentrations by the contact lens matrix.

It is noted that it is difficult to assess the effect of the PVAhydroxyl groups of the contact lens polymer on the boronic acid:sugarcomplexation reaction. However, studies performed with solutions ofglycerol indicated that monosaccharides have a greater binding affinityfor the novel phenyl boronic acid compounds than glycerol hydroxylgroups. As such, we speculate that the sugars will preferentially bindthe boronic acid groups of the BMQBA-doped contact lens.

It is also informative to consider the pH of tears as a potentialinterferent, given the response of these fluorophores to pH as shown inFIGS. 6 and 14. Unstimulated tear pH levels can vary in the range7.14-7.82 measured from healthy subjects at different times of the day,with a typical mean value around pH 7.45. However, a more acidic pH ofless than 7.3 is found following prolonged lid closure, e.g., aftersleep, which is thought to result from carbon dioxide produced by thecornea and trapped in the tear pool under the eyelids. While solutionsof these new fluorophores would be susceptible to these changes in pH,we have found that the doped lenses were unbufferable, hence externalchanges in pH are unlikely to affect the response to analytes.

6. Absorption and Emission Studies of Contact Lenses Containing BMOQBAin the Absence and Presence of Monosaccharides

FIG. 26 shows the leaching of o-BMOQBA from a BMOQBA-doped contact lensinto pH 7.5 buffer with time (see FIG. 26A), and the percent loss offluorescence intensity from the lens as a function of time (see FIG.26B). As discussed with reference to BMQBA, this experiment wasperformed simply to provide an indication of how long the lenses shouldbe leached before use. Based on FIG. 26B, the lenses were allowed toleach excess molecular sensing compound for 60 minutes before absorptionand emission measurements or the addition of sugars to the solutions.

Following leaching, buffered solutions of sugars are added to the lensholder. Because the 90% response time, which is the amount of timeneeded for the fluorescence signal to change by 90% of the initialvalue, was approximately 10 minutes, fluorescence spectra were typicallytaken approximately 15 minutes after each sugar addition to allow thelens to reach equilibrium.

FIGS. 27A and 27B show the fluorescence intensity of a o-BMOQBA-dopedcontact lenses with increasing concentrations of fructose and glucose,respectively, when the respective monosaccharides are injected into the1.5 cm³ contact lens volume. Similar to the solution based measurements(see FIGS. 12A and 12B), the molecular sensing moieties show a decreasein fluorescence intensity with increasing fructose and glucoseconcentration, which we attribute to the complexation of diols withboronic acid and subsequent charge neutralization. An intensity ratioplot, where I′ is the intensity in the absence of monosaccharide and Iis the intensity at the specified fructose (●) or glucose (▪)concentration, is shown in FIG. 28. As was observed with the solutionmeasurements, phenyl boronic acid derivatives seem to have a greateraffinity for fructose, which is based on the greater response offructose. However, referring to the insert of FIG. 28, when theconcentration of sugar is <1 mM, the response to both sugars wasidentical (Badugu, R., et al., J. Fluorescence, 13, 371-374 (2003)),with a 20% change in fluorescence signal with the addition of only 0.50mM sugar.

FIG. 29 shows the differences in response of the o-BMOQBA-doped contactlenses towards glucose relative to the solution-based studies (bothbuffered at pH 7.5).

Importantly, the response in the lens was greater than in the solutionsat the lower concentrations of glucose (see FIGS. 29B), which correspondto those concentrations normally observed in tears. It is unknown atthis time why at higher concentrations of glucose, the response shifts,whereby the greater response is observed in the solution-based studies.However, this shift is advantageous because the higher concentrations ofglucose, such as those observed in blood, can be readily monitored usinga solution-based measurement.

FIG. 30 shows the response of the halide sensitive o-BMOQBA fluorophorecontained within the contact lens to chloride ions, demonstrating thesensing capability of the fluorophore-doped contact lens to chloride,and hence bromide and iodide ions. Notably, the control compounds, e.g.,BMQ and BMOQ, which do not bind monosaccharides because of the lack of aboronic acid moiety, are equally sensitive to chloride ions. As such, itis envisioned that another alternative embodiment of this inventionutilizes both the novel phenyl boronic acid probes and the novel controlcompounds, e.g., by immobilizing one of each of the novel compounds inits own contact lens, to monitor monosaccharide and chloride levelssimultaneously. Alternatively, both compounds may be immobilized in thesame lens, and the spectral responses are simply resolved by the use ofdifferent excitation and emission wavelengths.

Monosaccharide lifetime sensing or ratiometric monosaccharide sensingare envisioned as embodiments employed to determine monosaccharideconcentrations using the novel phenyl boronic acid-doped contact lenses.

A further embodiment of this technology is the use of colored contactlenses which change color upon changes to the tear, and hence blood,glucose concentration.

7. Absorption and Emission Studies of Solutions Containing other BoronicAcid Fluorophores (BAFs) in the Absence and Presence of Monosaccharides

Stilbene, polyene and chalcone derivatives were tested to determinetheir suitability as monosaccharide-determining molecular sensingcompounds. Referring to FIG. 31, several boronic acid-containingfluorophores (BAFs) including: the stilbenes4′-dimethylaminostilbene-4-boronic acid (DSTBA) and4′-cyanostilbene-4-boronic acid (CSTBA); the polyene1-(p-boronophenyl)-4-(p-dimethylaminophenyl)buta-1,2-diene; and thechalcones3-[4′-(dimethylamino)phenyl]-1-(4′-boronophenyl)-prop-2-en-1-one(Chalc 1) and5-[4′-(dimethylamino)phenyl]-1-(4′-boronophenyl)-pent-2,4-dien-1-one(Chalc s) are shown. The preparation of the BAFs was in accordance withprevious reports (Dicesare, N., et al., Tetrahedron Letters, 43,2615-2618 (2002); Dicesare, N., et al., J. Biomed. Optics, 7(4), 538-545(2002)).

Each BAF was tested to determine the usefulness of these BAFs withregard to tear glucose sensing in a contact lens. Towards that end, bothsolution-based and doped contact lens-based measurements, the methods ofwhich are described above, were performed and subsequently compared.

Excited-state charge transfer (CT) mechanisms were exploited to inducespectral changes of the BAFs in the presence of monosaccharides. CT maybe applied because the boronic acid group (—B(OH)₂— electronwithdrawing) and an electron donor group are present on the samefluorophore. In the presence of monosaccharides and appropriate pHvalues, the boronic acid group is present in its anionic form

-   (represented by B or D in FIG. 1), and as such is no longer an    electron withdrawing group. Because the charge transfer nature of    the excited state has been perturbed by the presence of    monosaccharide and/or hydroxide ions, spectral changes can be    observed.

The stilbene DSTBA combines the electron withdrawing boronic acid groupwith the electron donating dimethylamino group. As such, in the presenceof monosaccharides, DSTBA is expected to demonstrate a reduced CT. Incontrast, CSTBA, which has two electron withdrawing groups—boronic acidand cyano—is expected to demonstrate an increased CT in the presence ofmonosaccharides.

FIG. 32A shows the effect of increasing concentrations of fructose onthe emission spectra of DSTBA in pH 8.0 buffer/methanol (2:1). It can beseen that as the concentration of fructose increases, the emissionspectra undergo a blue shift of about 30 nm (over a fructoseconcentration range of 0 mM→100 mM) and an increase in fluorescenceintensity. The blue shift and the change in intensity is attributed tothe reduction of electron withdrawing capability of the boronic acidmoiety with increasing monosaccharide complexation.

Ratiometrically, DSTBA demonstrates significant changes with increasingfructose and glucose concentrations (see FIG. 32C). For example, at 60mM sugar, fructose shows a 4.5-fold change in intensity, while glucoseshows a very respectable 2-fold change in intensity.

In contrast, the stilbene CSTBA, which includes two electron withdrawinggroups, demonstrated an emission spectra red shift of about 25 nm withincreasing fructose concentration (see FIG. 32B, which shows the effectof increasing concentrations of fructose on the emission spectra ofCSTBA in pH 8.0 buffer/methanol (2:1)), and a decrease in emissionintensity, which is opposite of that observed with DSTBA. It isspeculated that the red shift is due to an excited CT state caused bythe ionization of the boronate group with increasing sugarconcentrations, said boronate group acting as an electron donor group tothe cyano groups electron withdrawing capability.

Ratiometrically, CSTBA demonstrates significant changes with increasingfructose and glucose concentrations (see FIG. 32D), most notably forfructose.

FIG. 33A shows the effect of increasing concentrations of fructose onthe emission spectra of the polyene DDPBBA in pH 8.0 buffer/methanol(2:1). Like DSTBA, which also has an electron donating group (NMe₂), ablue shift was observed with increasing concentrations of sugar as wellas an increase in emission intensity.

Compounds that emit longer wavelength emission are particularlyattractive because (a) it reduces the detection of any lens or eyeautofluorescence as well as scatter, and (b) it allows one to usecheaper wavelength excitation sources, such as lasers and LEDs. Towardsthat end, the feasibility of using Chalc 1 and Chalc 2 fluorophores, wasinvestigated using compounds that were previously synthesized (Dicesare,N., et al., Tetrahedron Letters, 43, 2615-2618 (2002); Dicesare, N., etal., J. Biomed. Optics, 7(4), 538-545 (2002)). With chalcone derivativesthe charge transfer occurs between the dimethylamino group (electrondonating) and the carbonyl group (electron withdrawing). However, anychange to the electronic properties of the boron group leads to a changein the electron density of the benzophenone moiety and as such, thecharge transfer properties of the excited state of the fluorophore.Referring to FIG. 33B, which shows the effect of increasingconcentrations of fructose on the emission spectra (λ_(em)=665 nm) ofChalc 2 in pH 8.0 buffer/methanol (2:1), it can be seen that thechalcone derivative undergoes a blue shift and an increase in emissionintensity with increasing fructose concentrations. Chalc 1 showed acorresponding blue shift and increase in emission intensity with theemission centered around 580 nm.

8. Absorption and Emission Studies of Contact Lenses Containing otherBoronic Acid Fluorophores (BAFs) in the Absence and Presence ofMonosaccharides

As introduced above, prior to the addition of the sugars to the 1.5 cm³lens holder, the lenses were doped with the appropriate fluorophore,washed and allowed to leach for approximately 1 hour. Fluorescenceemissions were taken about 15 min after the addition of each aliquot ofsugar to the lens holder to allow the lens to reach equilibrium.

FIGS. 34A and 34B show the emission spectra of a DSTBA-doped contactlens in pH 8.0 buffer/methanol (2:1) with increasing concentrations ofglucose and fructose, respectively. As expected the intensity change inthe presence of fructose was greater. However, comparing the emissionresponse in the presence of fructose of DSTBA in the doped contact lens(see FIG. 34B) with those of the solution-based studies discussed above(see FIG. 32A), it can be seen that the responses were converse to oneanother, wherein the intensity of the emission increased with increasingfructose concentration in the solution-based experiment but decreased inthe corresponding doped contact lens experiment.

Similarly, FIGS. 34C and 34D show the emission spectra of a CSTBA-dopedcontact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of glucose and fructose, respectively. Comparing thefructose experiment performed in solution (see

FIG. 32B) with those of the doped contact lens (see FIG. 34D), it can beseen that the intensity similarly decreased with increasing fructoseconcentration. However, the significant red shift observed in thesolution-based studies was not observed in the doped contact lensstudies, suggesting that the electron-donating capability of the anionicboronate groups decreased.

FIG. 35 shows the simple intensity ratio of a (A) DSTBA-doped contactlens and (B) CSTBA-doped contact lens in pH 8.0 buffer/methanol (2:1) inthe absence, I′, and presence, I, of sugar. Interestingly, there is amore significant response to fructose in the CSTBA-doped contact lens(see FIG. 35B) than in the DSTBA-doped contact lens (see FIG. 35A).Regarding glucose, at concentrations of about 10 mM, there is a 10%fluorescence change in the DSTBA-doped contact lens, which is promising.

Comparing the emission spectra of DDPBBA-doped contact lenses in thepresence of fructose (see FIG. 36B) with those observed in thesolution-based experiments (see FIG. 33A), it can be seen that thoughthe blue shift similarly occurred in both with increasing fructoseconcentrations, the change in fluorescence intensity with increasingfructose concentrations was opposite, decreasing in the case of theDDPBBA-doped contact lens. It is noted that although this was similarlyobserved with respect of DSTBA, DSTBA displayed a more significantresponse to sugar (compare FIGS. 34B and 36B).

In light of the fact that the response to the sugars in the doped lenseswas opposite to that observed in solution, a series of emissionexperiments were performed using a DDPBBA solution in different pH mediain the presence of increasing glucose concentrations. As the solution pHincreases, the emission spectra of DDPBBA display a blue shift, whichcan be attributed to a change in acidity from the uncomplexed form atlow pH values (represented by A in FIG. 1) to the complexed form athigher pH values (represented by B in FIG. 1). Interestingly, thespectral response of the DDPBBA-doped contact lens in the presence ofglucose (see FIG. 36A) was similar to that observed in the pH 6.0 media(see FIG. 37D). It is speculated that at the lower pH, such as that ofthe contact lens, the formation of the boronic acid diester (representedby C in FIG. 1) does not result in a full perturbation of the DDPBBAfluorophore, hence DDPBBA is not suitable as a wavelength ratiometricprobe in the contact lens. It is noted that the DDPBBA fluorophore isideal for solution sugar sensing in the pH 6.5-8.5 range, which is idealto determine blood glucose levels.

Notably, BAF's such as DDPBBA are not fully perturbated at pH 6.0, andas such DDPBBA molecular sensing compounds are not able to sense glucoselevels in tears as effectively as the novel quaternary nitrogencompounds discussed herein which was the impetus for the discovery ofthe novel quaternary nitrogen containing boronic acid compounds,including, but not limited to, BMQBA and BMOQBA, which display lowerpK_(a) values and as such, are more sensitive to tear glucose levels.

FIGS. 38A and 38B show the emission spectra (λ_(em)=560 nm) of a Chalc2-doped contact lens in pH 8.0 buffer/methanol (2:1) with increasingconcentrations of (A) glucose and (B) fructose, respectively. It isnoted that the Chalc 1-doped contact lens displayed similar results tothe Chalc 2-doped contact lens, with the emission centered at λ=630 nm.Like DDPBBA and DSTBA, the emission intensity of the doped contact lensand the solution-based studies for both Chalc 1 and Chalc 2 wereopposite, decreasing in the case of the former. A simple intensity ratioof a Chalc 2-doped contact lens in pH 8.0 buffer/methanol (2:1) in theabsence, I′, and presence, I, of sugar is shown in FIG. 38C.Interestingly, the solution response for Chalc 2 in the presence of 100mM fructose at pH 8.0 produces a 3-fold increase in fluorescenceemission (see FIG. 33B), as compared to the approximately 2.6-foldreduction for the same fructose concentration in the contact lens.

9. Compounds (L), (M) and (N) as Glucose-Sensing Molecules for CovalentBinding to Contact Lenses

Compounds (L), (M), (N) and (O), previously described herein, weresynthesized. Compound (O) was utilized as a control compound, devoid ofboronic acid functionality and therefore unable to bind and senseglucose, to assess the di-boronic acid compounds (L) and (M) and themono-boronic acid compound (N).

The di-boronic acid compounds (L) and (M) were utilized with theintention of achieving glucose specificity in contact lenses, withmolecules in which the spacing distance between the boronic acid groupswas determined to be specific for interaction with glucose. Moleculeshaving glucose specificity in the presence of other sugars orcarbohydrates are particularly preferred compounds, as being free ofinterference for such glucose sensing.

FIGS. 39-42 show the response of the di-boronic acid probes, compound(L), denoted p-DBQBA, and compound (M), denoted o-DBQBA, towardsglucose. The probes were immobilized in a contact lens by covalentbonding through the reactive alkenyl functionality of such compounds.

FIG. 39 shows the emission spectra (λ_(ex)=360 nm) of compound (L) in pH7.0 buffer with glucose. FIG. 40 shows the intensity ratio for responseof compound (L) towards sugars in pH 7.0 phosphate buffer.

FIG. 41 shows the emission spectra (λ_(ex)=360 nm) of compound (M) in pH7.0 buffer with glucose. FIG. 42 shows the intensity ratio for responseof compound (M) towards sugars in pH 7.0 buffer.

Interestingly, the glucose binding constants of these probes was about2-3 times lower as compared to o-N-(boronobenzyl)-6-methoxyquinoliniumbromide, which had a Kd of about 180 mM. This was attributed to sterichindrance effects and the lower diffusion rate of glucose in the lens ascompared to free solution, as well as the matrix effect in the lens, inwhich some probe molecules are inaccessible to glucose after covalentattachment.

To further assess this response behavior, compound (N), denoted o-MBQBA,having only a single boronic acid group, was evaluated, yielding theresults shown in FIGS. 43 and 44.

FIG. 43 shows the emission spectra (λ_(ex)=345 nm) of compound (N) in pH7.0 buffer with glucose. FIG. 44 shows the intensity ratio for responseof compound (N) towards sugars in pH 7.0 buffer.

When the glucose response is compared for both ortho compounds (M) and(N) when bound into the contact lens, the mono-boronic acid probe(compound (N)) exhibited slightly better response, suggesting thatbinding of glucose to both boronic acid groups in the di-boronic acidspecies did not occur with the same probability and that after binding,steric hindrance effects impeded further glucose binding.

All of the boronic acid-functionalized compounds (L)-(N) thusdemonstrated glucose sensing capability for physiological tear levels inthe mildly acid and modestly polar medium of the contact lens.

While the invention has been described herein with reference to variousspecific embodiments, it will be appreciated that the invention is notthus limited, and extends to and encompasses various other modificationsand embodiments, as will be

1. A fluorescence compound selected from the group consisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups.
 2. A fluorescence compound having the structure:

wherein X is chloride, bromide or iodide, and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups.
 3. The compound of claim 2, wherein X is bromide and R is —CH₃,—OCH₃ or —NH₂.
 4. The compound of claim 2, wherein R is —CH₃.
 5. Thecompound of claim 2, wherein R is —OCH₃.
 6. The compound of claim 2,wherein R is NH₂.
 7. The compound of claim 2, wherein the fluorescenceintensity of the compound changes in the presence of analytes.
 8. Thecompound of claim 7, wherein the analyte comprise a species selectedfrom the group consisting of glucose, fructose, chloride and iodide. 9.The compound of claim 2, wherein the fluorescence intensity of thecompound decreases in the presence of glucose.
 10. A method of measuringthe concentration of an analyte in a physiological fluid, said methodcomprising: (a) contacting a fluorescence compound selected from thegroup consisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups, with the physiological fluid for sufficient time to at leastpartially interact or react with the analyte; and (b) measuring theoptical signal of the fluorescence compound in the presence of theanalyte for a sufficient time to determine the concentration of analytein the physiological fluid.
 11. The method of claim 10, wherein thephysiological fluid is blood, tears, interstitial fluid or aqueoushumor.
 12. The method of claim 10, wherein the measuring of the analyteis continuous.
 13. The method of claim 10, wherein the physiologicalfluid is tears.
 14. The method of claim 10, wherein the fluorescencecompound is


15. The method of claim 14, wherein X is bromide and R is —CH₃, —OCH₃ or—NH₂.
 16. The method of claim 15, wherein R is —CH₃.
 17. The method ofclaim 15, wherein R is —NH₂.
 18. The method of claim 10, wherein theanalyte comprises a species selected from the group consisting ofglucose, fructose, chloride and iodide.
 19. The method of claim 10,wherein the analyte comprises glucose.
 20. The method of claim 10,wherein the optical signal is a fluorescence signal.
 21. The method ofclaim 20, wherein the fluorescence is measured based on the intensity,lifetime, anisotropy or ratiometric fluorescence platforms.
 22. Themethod of claim 20, wherein the concentration of the analyte isdetermined by comparing the fluorescence measurement in the presence ofanalyte with the fluorescence measurement in the absence of analyte. 23.The method of claim 20, wherein the concentration of the analyte isdetermined by comparing the fluorescence measurement in the presence ofanalyte with the fluorescence measurement in the presence of a knownconcentration of analyte.
 24. The method of claim 10, wherein thefluorescence compound is in solution.
 25. The method of claim 10,wherein the fluorescence compound is contained in an analyte-permeablematrix.
 26. An assay kit for measuring the concentration of an analytein a physiological fluid comprising a container containing afluorescence compound selected from the group consisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups.
 27. The kit of claim 26, wherein the physiological fluid isblood.
 28. The kit of claim 26, wherein the analyte comprises glucose.29. The kit of claim 26, wherein the fluorescence compound is:


30. The kit of claim 29, wherein X is bromide and R is —CH₃, —OCH₃ or—NH₂.
 31. An ophthalmic sensor comprising: a polymer matrix comprising afluorescence compound contacting at least the outer surfaces of thepolymer matrix, wherein the fluorescence compound interacts or reactswith an analyte to provide an optical signal which is indicative of theanalyte concentration in an ocular fluid and wherein the fluorescencecompound is at least one member selected from the group consisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups;


32. The ophthalmic sensor of claim 31, wherein the fluorescence compoundis:


33. The ophthalmic sensor of claim 32, wherein X is bromide and R is—CH₃, —OCH₃ or —NH₂.
 34. The ophthalmic sensor of claim 31, wherein theophthalmic device is a contact lens.
 35. The ophthalmic sensor of claim34, wherein the fluorescence compound is coated on the surface of acontact lens.
 36. The ophthalmic sensor of claim 34, wherein thefluorescence compound is impregnated into the polymer matrix of whichthe contact lens is fabricated thereof.
 37. The ophthalmic sensor ofclaim 36, wherein the contact lens is disposable.
 38. The ophthalmicsensor of claim 31, wherein the fluorescence compound is covalentlyattached to the surface of the ophthalmic device.
 39. The ophthalmicsensor of claim 31, wherein the fluorescence compound is physicallybonded to the surface of the ophthalmic device.
 40. The ophthalmicsensor of claim 31, wherein the ophthalmic device is implantable. 41.The ophthalmic sensor of claim 31, wherein the analyte comprises aspecies selected from the group consisting of glucose, fructose,chloride, and iodide.
 42. The ophthalmic sensor of claim 31, wherein theanalyte comprises glucose.
 43. The ophthalmic sensor of claim 31,wherein the optical signal is a fluorescence signal.
 44. The ophthalmicsensor of claim 43, wherein the fluorescence is measured based on theintensity, lifetime, anisotropy or ratiometric fluorescence platforms.45. A method of measuring the concentration of glucose in ocular fluid,said method comprising: (a) contacting a fluorescence compound selectedfrom the group consisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups; and with the ocular fluid for sufficient time to at leastpartially interact or react with the glucose to provide an opticalsignal which is indicative of the analyte concentration in an ocularfluid.
 46. The method of claim 45, wherein the optical signal decreaseswith the increased concentration of glucose.
 47. The method of claim 45,wherein the fluorescence compound is:


48. The method of claim 47, wherein X is bromide and R is —CH₃, —OCH₃ or—NH₂.
 49. The method of claim 45, wherein the concentration of glucoseis in a range from about 50 uM to about 5 mM.
 50. The method of claim45, wherein the optical signal is a fluorescence signal.
 51. The methodof claim 50, wherein the fluorescence is measured based on theintensity, lifetime, anisotropy or ratiometric fluorescence platforms.52. The method of claim 50, wherein the concentration of the glucose isdetermined by comparing the fluorescence measurement in the presence ofglucose with the fluorescence measurement in the absence of glucose. 53.The method of claim 50, wherein the concentration of the glucose isdetermined by comparing the fluorescence measurement in the presence ofglucose with the fluorescence measurement in the presence of a knownconcentration of glucose.
 54. The method of claim 45, wherein thefluorescence compound is coated on a contact lens.
 55. The method ofclaim 54, wherein the contact lens is disposable.
 56. The method ofclaim 54, further comprising a second fluorescence compound that issensitive to chloride.
 57. An analyte sensor comprising: a substratecomprising a fluorescence compound contacting at least the outersurfaces of the substrate, wherein the fluorescence compound interactsor reacts with an analyte to provide an optical signal which isindicative of the analyte concentration in a physiological fluid andwherein the fluorescence compound is at least one member selected fromthe group consisting of:

wherein X is chloride, bromide or iodide and R is selected from thegroup consisting of H, straight chain or branched C₁-C₄ alkyl group,C₁-C₄ alkoxy group, aryl group, hydroxyl, cyano, sulfonyl, and NR¹R²,wherein R¹ and R² may be the same as or different from one another andis independently selected from the group consisting of H and C₁-C₄ alkylgroups.
 58. The analyte sensor of claim 57, wherein the fluorescencecompound is:


59. The analyte sensor of claim 58, wherein X is bromide and R is —CH₃,—OCH₃ or —NH₂.
 60. The analyte sensor of claim 57, wherein the substrateis a contact lens.
 61. The analyte sensor of claim 57, wherein thesubstrate is implantable.
 62. The analyte sensor of claim 57, whereinthe analyte comprises a member selected from the group consisting ofglucose, fructose, chloride, and iodide.
 63. The analyte sensor of claim57, wherein the optical signal is fluorescence and is measured based onthe intensity, lifetime, anisotropy or ratiometric fluorescenceplatforms.
 64. A compound selected from the group consisting of:


65. An ophthalmic device including a substrate having at least onecompound according to claim 64 covalently bonded thereto.
 66. Theophthalmic device of claim 65, comprising a contact lens.
 67. An analytesensor comprising a heterocyclic quaternary nitrogen compound containingat least one heterocyclic quaternary ring nitrogen that is linkedthrough a phenyl ring with a boronic acid group —B(OH)₂.
 68. The analytesensor of claim 67, wherein the heterocyclic quaternary nitrogencompound comprises a benzyl group pendant from the heterocyclicquaternary ring nitrogen and having a boronic acid substituent on aphenyl ring of the benzyl group.
 69. The analyte sensor of claim 67,wherein the heterocyclic quaternary nitrogen compound is covalentlybonded to a support.
 70. The analyte sensor of claim 69, wherein thesupport comprises a polymeric medium.
 71. The analyte sensor of claim67, wherein the heterocyclic quaternary nitrogen compound includesmultiple heterocyclic quaternary ring nitrogens, each having a benzylmoiety bonded thereto, with boronic acid substituents on one or morephenyl rings of the benzyl moieties.
 72. A method of determining levelof an analyte at a locus containing or susceptible to presence of saidanalyte, said method comprising exposing to said locus an analyte sensoraccording to claim 67, and determining from an optical fluorescencesignal of said heterocyclic quaternary nitrogen compound the level ofsaid analyte at said locus.